Methods of treatng B-cell diseases using humanized anti-CD19 antibodies

ABSTRACT

The present invention provides humanized, chimeric and human anti-CD19 antibodies, anti-CD19 antibody fusion proteins, and fragments thereof that bind to a human B cell marker. Such antibodies, fusion proteins and fragments thereof are useful for the treatment and diagnosis of various B-cell disorders, including B-cell malignancies and autoimmune diseases.

BACKGROUND OF THE INVENTION

This application is a divisional to U.S. patent application Ser. No.10/903,858 (now issued U.S. Pat. No. 7,109,304), “Humanized Anti-CD19Antibodies” filed on Aug. 22, 2004, which claims priority to aprovisional U.S. Patent Application No. 60/491,282, filed Jul. 31, 2003,the contents of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to anti-CD19 antibodies, particularlyhumanized, chimeric and human anti-CD19 antibodies, particularlymonoclonal antibodies (MAbs) and fragments thereof, either naked orconjugated to at least one therapeutic and/or diagnostic agent, andmethods of use thereof. In particular, the anti-CD19 antibodies of thepresent invention can be used for treating B cell disease such as, forexample, a malignancy and/or an inflammatory disease or disorder,including an autoimmune disease. The methods and compositions of thepresent invention can also be used to treat a lymphoma and a leukemia,including non-Hodgkin's lymphoma, chronic lymphocytic leukemia, andacute lymphoblastic leukemia. In a preferred embodiment, the neoplasticdisorder is a B-cell malignancy such as indolent forms of B-celllymphomas, aggressive forms of B-cell lymphomas (including non-Hodgkin'slymphoma), chronic lymphatic leukemias, or acute lymphatic leukemias, ormultiple myeloma.

The present invention also relates to antibody fusion proteins andfragments thereof comprising at least two anti-CD19 MAbs or fragmentsthereof, or at least one anti-CD19 MAb or fragment thereof and at leastone second MAb or fragment thereof, other than the anti-CD19 Mab orfragment thereof. The multispecific and/or fusion proteins of thepresent invention can be either naked or conjugated to at least onetherapeutic and/or diagnostic agent.

The humanized, chimeric and human anti-CD19 Mabs and fragments thereof,and antibody fusion proteins and fragments thereof may be administeredalone, either naked or conjugated to a therapeutic or diagnostic agent,or in combination with another naked antibody, fragment orimmunoconjugate. Also, naked or conjugated anti-CD 19 antibodies andfragments thereof, and antibody fusion proteins and fragments thereofmay be administered in combination with at least one therapeutic agentor diagnostic agent that is not conjugated to a CD 19 antibody orfragment thereof, or fusion protein or fragment thereof.

Additionally, the present invention relates to a DNA sequence encoding ahumanized, chimeric or human anti-CD19 antibody and fragment thereof,and antibody fusion protein and fragment thereof. Likewise, a vector andhost cell containing the DNA sequence is also contemplated. Finally, thepresent invention discloses methods of making the humanized, chimericand human anti-CD 19 antibodies and fragments thereof, and fusionproteins and fragments thereof.

BACKGROUND

The immune system of vertebrates consists of a number of organs and celltypes which have evolved to accurately recognize foreign antigens,specifically bind to, and eliminate/destroy such foreign antigens.Lymphocytes, among other cell types, are critical to the immune system.Lymphocytes are divided into two major sub-populations, T cells and Bcells. Although inter-dependent, T cells are largely responsible forcell-mediated immunity and B cells are largely responsible for antibodyproduction (humoral immunity).

In humans, each B cell can produce an enormous number of antibodymolecules. Such antibody production typically ceases (or substantiallydecreases) when a foreign antigen has been neutralized. Occasionally,however, proliferation of a particular B cell will continue unabated andmay result in a cancer known as a B cell lymphoma. B-cell lymphomas,such as the B-cell subtype of non-Hodgkin's lymphoma, are significantcontributors to cancer mortality. The response of B-cell malignancies tovarious forms of treatment is mixed. For example, in cases in whichadequate clinical staging of non-Hodgkin's lymphoma is possible, fieldradiation therapy can provide satisfactory treatment. Still, aboutone-half of the patients die from the disease. Devesa et al., J. Nat'lCancer Inst. 79:701 (1987).

The majority of chronic lymphocytic leukemias are of the B-cell lineage.Freedman, Hematol. Oncol. Clin. North Am. 4:405 (1990). This type ofB-cell malignancy is the most common leukemia in the Western world.Goodman et al., Leukemia and Lymphoma 22: 1 (1996). The natural historyof chronic lymphocytic leukemia falls into several phases. In the earlyphase, chronic lymphocytic leukemia is an indolent disease,characterized by the accumulation of small maturefunctionally-incompetent malignant B-cells having a lengthened lifespan. Eventually, the doubling time of the malignant B-cells decreasesand patients become increasingly symptomatic. While treatment canprovide symptomatic relief, the overall survival of the patients is onlyminimally affected. The late stages of chronic lymphocytic leukemia arecharacterized by significant anemia and/or thrombocytopenia. At thispoint, the median survival is less than two years. Foon et al., AnnalsInt. Medicine 113:525 (1990). Due to the very low rate of cellularproliferation, chronic lymphocytic leukemia is resistant to cytotoxicdrug treatment.

Traditional methods of treating B-cell malignancies, includingchemotherapy and radiotherapy, have limited utility due to toxic sideeffects. The present invention minimizes drug toxicity of normal tissuesby using conjugated monoclonal antibodies and antibody fragments toselectively direct a radionuclide, toxin, RNAi molecule or othertherapeutic or diagnostic agent to a tumor site. In addition,unconjugated B-cell antibodies, such as anti-CD 19, -CD20, -CD21, -CD23,-CD80 and -CD22 antibodies can be used to target certain markers onB-cell malignancies. Also, other antigens, such as HLA-DR may serve astargets for both normal and malignant B-cells, even though they are alsoexpressed on other cell types.

B cells comprise cell surface proteins which can be utilized as markersfor differentiation and identification. One such human B-cell marker isa CD19 antigen and is found on mature B cells but not on plasma cells.CD19 is expressed during early pre-B cell development and remains untilplasma cell differentiation. CD19 is expressed on both normal B cellsand malignant B cells whose abnormal growth can lead to B-celllymphomas. For example, CD19 is expressed on B-cell lineagemalignancies, including, but not limited to non-Hodgkin's lymphoma,chronic lymphocytic leukaemia, and acute lymphoblastic leukaemia.

A potential problem with using non-human monoclonal antibodies (e.g.,murine monoclonal antibodies) is typically lack of human effectorfunctionality. In other words, such antibodies may be unable to mediatecomplement-dependent lysis or lyse human target cells throughantibody-dependent cellular toxicity or Fc-receptor mediatedphagocytosis. Furthermore, non-human monoclonal antibodies can berecognized by the human host as a foreign protein and, therefore,repeated injections of such foreign antibodies can lead to the inductionof immune responses leading to harmful hypersensitivity reactions. Formurine-based monoclonal antibodies, this is often referred to as a HumanAnti-Mouse Antibody (HAMA) response.

The use of chimeric antibodies is more preferred because they do notelicit as strong a HAMA response as murine antibodies. Chimericantibodies are antibodies which comprise portions from two or moredifferent species. For example, Liu, A. Y. et al, “Production of aMouse-Human Chimeric Monoclonal Antibody to CD20 with PotentFc-Dependent Biologic Activity” J. Immun. 139/10:3521-3526 (1987),describe a mouse/human chimeric antibody directed against the CD20antigen. See also, PCT Publication No. WO 88/04936. However, noinformation is provided as to the ability, efficacy or practicality ofusing such chimeric antibodies for the treatment of B cell disorders inthe reference. It is noted that in vitro functional assays (e.g.,complement-dependent lysis (CDC); antibody dependent cellularcytotoxicity (ADCC), etc.) cannot inherently predict the in vivocapability of a chimeric antibody to destroy or deplete target cellsexpressing the specific antigen. See, for example, Robinson, R. D. etal., “Chimeric mouse-human anti-carcinoma antibodies that mediatedifferent anti-tumor cell biological activities,” Hum. Antibod.Hybridomas 2:84-93 (1991) (chimeric mouse-human antibody havingundetectable ADCC activity). Therefore, the potential therapeuticefficacy of a chimeric antibody can only truly be assessed by in vivoexperimentation, preferably in the species of interest for the specifictherapy.

One approach that has improved the ability of murine monoclonalantibodies to be effective in the treatment of B-cell disorders has beento conjugate a radioactive label or chemotherapeutic agent to theantibody, such that the label or agent is localized at the tumor site.For example, studies indicate that ⁹⁰Y labeled anti-CD19 antibodies canbe used to reduce lymphoma in mice (McDevitt et al., Leukemia 16:60(2002>>, anti-CD19 antibodies conjugated to idarubicin result in tumorregression in an experimental model (Rowland et al., Cancer Immunol.Immunother., 37:195 (1993)), and ¹²⁵I and ¹¹¹In radiolabeled CD19 isspecifically taken up in tumor bearing organs (Mitchell et al., J. Nucl.Med., 44: 1105 (2003)). Combination therapy with an anti-CD19 antibodyis also described in Ek et al., Leuk. Lymphoma 31: 143 (1998) and Uckumet al., Blood, 79:3116 (1992). Treatment of human B cell lymphoma withwith an anti-CD19 antibody and CD3×CD19 diabody is described in Hekmanet al., Cancer Immunol. Immunother., 32:364 (1991) and Cochlovius etal., J. Immunol., 165:888 (2000), respectively.

However, these approaches have not eliminated the obstacles associatedwith using murine antibodies, despite the fact that many patients withlymphoma who have received prior aggressive cytotoxic chemotherapy areimmune suppressed, thus having lower HAMA rates than lymphoma patientswho have not been heavily pretreated.

Inflammatory diseases, including autoimmune diseases are also a class ofdiseases associated with B-cell disorders. Examples of autoimmunediseases include, but are not limited to acute idiopathicthrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura,dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcalnephritis, erythema nodosurn, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitisubiterans, Sjogren's syndrome, primary biliary cirrhosis,Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic activehepatitis, polymyositis/dermatomyositis, polychondritis, parnphigusvulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophiclateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia,perniciousanemia, rapidly progressive glomerulonephritis, psoriasis, andfibrosing alveolitis. The most common treatments are corticosteroids andcytotoxic drugs, which can be very toxic. These drugs also suppress theentire immune system, can result in serious infection, and have adverseaffects on the bone marrow, liver and kidneys. Other therapeutics thathave been used to treat Class III autoimmune diseases to date have beendirected against T-cells and macrophages. There is a need for moreeffective methods of treating autoimmune diseases, particularly ClassIII autoimmune diseases.

To address the many issues related to B-cell disorders and theirtreatment, the present invention provides humanized, chimeric and humananti-CD19 monoclonal antibodies and fragments thereof, and antibodyfusion proteins and fragments thereof for the treatment of B celllymphomas and leukemias and autoimmune disorders in humans and othermammals without the adverse responses associated with using murineantibodies. The antibodies, fusion proteins and fragments thereof of thepresent invention can be used alone, conjugated to at least onediagnostic and/or therapeutic agent or in combination with othertreatment modalities.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides humanized, chimeric andhuman anti-CD19 antibodies that bind to a human B cell marker, referredto as CD19, which is useful for the treatment and diagnosis of B-celldisorders, such as B-cell malignancies and autoimmune diseases.

The present invention further provides methods of treatment of mammaliansubjects, such as humans or domestic animals, with one or morehumanized, chimeric or human CD19 antibodies, alone, as an antibodyfusion protein, as a therapeutic conjugate alone or as part of anantibody fusion protein, in combination, or as a multimodal therapy,with other antibodies, other therapeutic agents or immunomodulators oras an immunoconjugate linked to at least one therapeutic agent,therapeutic radionuclide or immunomodulator. These humanized, chimericand human CD19 antibodies can also be used as a diagnostic imaging agentalone, in combination with other diagnostic imaging agents, and/or inconjunction with therapeutic applications.

The present invention additionally is directed to anti-CD19 MAbs orfragments thereof that contain specific murine CDRs or a combination ofmurine CDRs from more than one murine or chimeric anti-CD19 MAb thathave specificity for CD19. These MAbs can be humanized, chimeric orhuman anti-CD19 MAbs.

The present invention is also directed to antibody fusion proteinscomprising at least two anti-CD19 MAbs or fragments thereof or a firstMAb comprising an anti-CD19 MAb or fragment thereof and a second MAb.

The present invention is further directed to a therapeutic or diagnosticconjugate of the anti-CD19 MAbs or fragments thereof and antibody fusionproteins of the anti-CD19 MAbs or other MAbs or fragments thereof boundto at least one therapeutic agent and/or at least one diagnostic agent.Antibody fusion proteins with multiple therapeutic agents of the same ordifferent type are also contemplated in the present invention.

The present invention is additionally directed to a method of using theanti-CD19 MAbs or fragments thereof or antibody fusion proteins thereofor fragments thereof for therapy, either alone, in combination with eachother, naked, conjugated to one or more therapeutic agents or eachadministered in combination with one or more therapeutic agents.

The present invention further is directed to a method of using theanti-CD19 MAbs or fragments thereof or antibody fusion proteins thereofor fragments thereof as a diagnostic bound to one or more diagnosticagents.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 discloses the V_(K), the variable light chain, and the VH, thevariable heavy chain, sequences of cA19, a chimeric anti-CD 19 antibody.The CDR region sequences are shown in bold and underlined. Thenucleotides are numbered sequentially. Kabat's Ig molecule numberi ngisused for amino acid residues as shown by the numbering above the aminoacid residues. The amino acid residues numbered by letters are theinsertion residues defined by Kabat numbering scheme. The insertionresidues have the same preceding digits as that of the pre viousresidue. Forexample, residues 82, 82A, 82B, and 82C in FIG. 1B areindicated as 82, A, B, and C, respectively. The light chain variableregion is shown in FIG. 1A (SEQ ID NO:1 and SEQ ID NO:2) and the heavychain variable region is shown in FIG. 1B (SEQ ID NO:3 and SEQ ID NO:4).

FIG. 2 shows the results of cell surface competitive binding assay tocompare the binding specificity of the cA19 antibody with that of otheranti-CD19 antibodies, BU12 and B4. Increasing concentrations of cA19blocked the binding of radiolabled BU12 to Raji cells in a similarfashion as the unlabeled BU12 and B4, indicating these antibodiesrecognize similar or overlap epitopes of CD19 molecule.

FIG. 3 compares the amino acid sequences of the variable light chain(Vk) and variable heavy chain (VH) of human antibodies, the chimeric andhumanized anti-CD19 antibodies. FIG. 3A compares the amino acidsequences of the variable light chain (Vk) of the human antibody,(REIVk; SEQ ID NO:5), a chimeric antibody, (cA19Vk; SEQ ID NO:6), and ahumanized antibody, (hA19Vk; SEQ ID NO:7), and FIG. 3B compares theamino acid sequences of the variable heavy chain (VH) of the humanantibodies, EU (SEQ ID NO:5) and NEWM (FR4 only; SEQ ID NO:11), thechimeric antibody, (cA19VH; SEQ ID NO:9) and a humanized antibody(hA19VH; SEQ ID NO:10).

FIG. 4 discloses the amino acid sequences of the humanized anti-CD19antibody, hA19, light chain V gene, (hA19Vk) (FIG. 4A; SEQ ID NO:12 andSEQ ID NO:13), and heavy chain Vgene, hA19VH (FIG. 4B; SEQ ID NO:14 andSEQ ID NO:15). The nucleotide sequences are shown in lower case.Numbering of Vk and VH amino acid residues is same as that in FIG. 1.

FIG. 5 shows the results of cell surface competitive binding assay tocompare the binding specificity and activity of the humanized A19antibody, hA19, with that of cA19. FIG. 5A shows both unconjugated hA19(closed triangles) and cA19 (closed circles) blocked the binding of¹²⁵I_hA19 to Raji cells. FIG. 5B shows hA19 (closed circles) and c19(closed squares) competed equally well for the binding of ¹²⁵I_cA19 toRaji cells. Increasing concentrations of either cA19 or hA19 blocked thebinding of radiolabled hA19 or cA19 to Raji cells respectively.

FIG. 6 shows the determination of the Ag-binding affinity (avidity) ofthe anit-CD19 Ab by the direct cell surface binding and Scatchard plotanalysis. Varying concentrations of ¹²⁵I-hA19 (diamonds) or ¹¹²I-cA19(squares) were incubated with Raji cells at ⁴⁰ C for 1 h. Total andbound radio activities were counted and analyzed by Scatchard plot asshown in the inset. hA19 showed virtually same binding affinity as cA19.As shown the apparent dissociation constant values were calculated to be1.1 and 1.2 nM for hA19 and cA19, respectively.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise specified, “a” or “an” as used herein means “one ormore.”

1. Overview

As discussed above, anti-CD19 antibodies that are unconjugated orlabeled with a therapeutic radionuclide, have failed to provide highrates of objective and lasting responses in patients with intermediateor aggressive forms of B-cell lymphoma. The present invention provides ahumanized, a chimeric and a human anti-CD19 antibody and antibody fusionproteins thereof useful for treatment of mammalian subjects, humans anddomestic animals, alone, as a conjugate or administered in combinationwith other therapeutic agents, including other naked antibodies andantibody therapeutic conjugates.

The anti-CD19 MAbs of the present invention contain specific murine CDRsor acombination of murine CDRs from more than one murine or chimericanti-CD19 MAb that have specificity for the CD19 antigen. The anti-CD19MAbs of the present invention are humanized, chimeric or human MAbs andthey contain the amino acids of the CDRs of a murine anti-CD19 MAb andthe light and heavy chain constant regions of a human antibody, whileretaining substantially the B-cell and B-cell lymphoma and leukemia celltargeting of the murine anti-CD19 MAb. The CDRs of the light chainvariable region of the anti-CD19 MAb comprises CDR1 comprising aminoacids KASQSVDYDGDSYLN (SEQ ID NO: 16); CDR2 comprising amino acidsDASNLVS (SEQ ID NO: 17); and CDR3 comprising amino acids QQSTEDPWT (SEQIDNO: 18); and the CDRs of the heavy chain variable region of theanti-CD19 MAb comprises CDR1 comprising amino acids SYWMN (SEQ ID NO:19); CDR2 comprising amino acids QIWPGDGDTNYNGKFKG (SEQ ID NO: 20) andCDR3 comprising amino acids RETTTVGRYYYAMDY (SEQ ID NO: 21).

In a preferred embodiment, the humanized anti-CD19 MAb or fragmentthereof of the present invention comprises the CDRs of a murineanti-CD19 MAb and the framework (FR) regions of the light and heavychain variable regions of a human antibody and the light and heavy chainconstant regions of a human antibody, while retaining substantially theB-cell, and B-cell lymphoma and leukemia cell targeting of the parentmurine anti-CD19 MAb, and wherein the CDRs of the light chain variableregion of the anti-CD 19 MAb comprises CDR1 comprising amino acidsKASQSVDYDGDSYLN (SEQ ID NO: 16); CDR2 comprising amino acids DASNLVS(SEQ ID NO: 17); and CDR3 comprising amino acids QQSTEDPWT (SEQ IDNO:18); and the CDRs of the heavy chain variable region of the anti-CD19MAb comprises CDR1 comprising amino acids SYWMN (SEQ ID NO: 19); CDR2comprising amino acids QIWPGDGDTNYNGKFKG (SEQ ID NO: 20) and CDR3comprising amino acids RETTTVGRYYYAMDY (SEQ ID NO: 21). But thehumanized anti-CD19 MAb or fragment thereof may further contain in theFRs of the light and heavy chain variable regions of theantibody atleast one amino acid from the corresponding FRs of the murine MAb.Specifically, the humanized anti-CD 19 MAb or fragment thereof containsat least one amino acid residue 5, 27, 28, 40, 48, 91, 94, 107, and 108of the murine heavy chain variable region of FIG. 4A, designated ashA19VH (SEQ ID NO:10) and of at least one amino acid residue 4, 39, 58,60, 87, 100, and107 of the murine light chain variable region FIG. 4B,designated hA19Vk (SEQ ID NO:13). Oneor more of the murine amino acidsequences can be maintained in the human FR regions of the light andheavy variable chains if necessary to maintain proper binding or toenhance binding tothe CD 19 antigen. More preferably the humanizedanti-CD19 MAb or fragment thereof of the present invention comprises thehA19Vk (SEQ ID NO:13) of FIG. 3A and the hA19VH FIG. 3B(SEQ ID NO:10).

The preferred chimeric anti-CD19 (cA19) MAb or fragment thereof of thepresent invention comprises the CDRs of a murine anti-CD19 MAb and theFR regions of the light and heavy chain variable regions of the murineanti-CD 19 MAb, i.e., the Fvs of the parental murine MAb, and the lightand heavy chain constant regions of a human antibody, wherein thechimeric anti-CD 19 MAb or fragment thereof retains substantially theB-cell, and B-cell lymphoma and leukemia cell targeting of the murineanti-CD 19 MAb, wherein the CDRs of the light chain variable region ofthe anti-CD19 MAb comprises CDR1 comprising amino acids KASQSVDYDGDSYLN(SEQ ID NO: 16); CDR2 comprising amino acids DASNLVS (SEQ IDNO: 17); andCDR3 comprising amino acids QQSTEDPWT (SEQ ID NO: 18); and the CDRs ofthe heavy chain variable region of the anti-CD 19 MAb comprises CDR1comprising amino acids SYWMN (SEQ ID NO: 19); CDR2 comprising aminoacids QIWPGDGDTNYNGKFKG (SEQID NO:20) and CDR3 comprising amino acidsRETTTVGRYYYAMDY (SEQ ID NO: 21).

More preferably the chimeric anti-CD19 MAb or fragment thereofcomprising the complementarity-determining regions (CDRs) of a murineanti-CD19 MAb and the framework(FR) regions of the light and heavy chainvariable regions of the murine anti-CD 19 MAb and the light and heavychain constant regions of a human antibody, wherein the chimericanti-CD19 MAb or fragment thereof retains substantially the B-cell, andB-cell lymphoma and leukemia cell targeting of the murine anti-CD19 MAb,wherein the CDRs of the light chain variable region of the chimericanti-CD19 MAb comprises the CDRs shown in FIGS. 2A and 2B, respectively,designated cA19Vk (SEQ ID NO:6) and cA19VH (SEQ ID NO:9).

The present invention also encompasses a human anti-CD19 MAb or fragmentthereof comprising the light and heavy chain variable and constantregions of a human antibody, wherein said human anti-CD19 MAb retainssubstantially the B-cell, and B-cell lymphoma and leukemia celltargeting and cell binding characteristics of a murine anti-CD19 MAb,wherein the CDRs of the light chain variable region of the humananti-CD19 MAb comprises the same CDRs as set forth above for thechimeric and humanized anti-CD19 MAbs and as shown in FIGS. 2A and 2B,and 3A and 3B, respectively.

The present invention is also intended to encompass antibody fusionproteins or fragments thereof comprising at least two anti-CD19 MAbs orfragments thereof, as described above. The antibody fusion protein orfragment thereof of the present invention is also intended to encompassan antibody fusion protein or fragment thereof comprising at least onefirst anti-CD19 MAb or fragment thereof as described above and at leastone second MAb or fragment thereof, other than the anti-CD19 MAb orfragment described above. More preferably this second MAb is a MAbreactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23,CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80,CD126, CD138, B7, MUC-1, Ia, HM1.24, HLA-DR, tenascin, an angiogenesisfactor, VEGF, PIGF, ED-B fibronectin, an oncogene, an oncogene product,NCA 66a-d, necrosis antigens, Ii, IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4)and TRAIL-R2 (DR5) or a combination thereof, and even an anti-CD19 MAbthat is directed to a different epitope than the anti-CD19 MAb describedherein. The antibody fusion proteins of the present invention may becomposed of one CD19 MAb and one or more of the second MAbs to providespecificity to different antigens, and are described in more detailbelow.

The humanized, chimeric and human anti-CD19 antibody may possessenhanced affinity binding with the epitope, as well as antitumor andanti-B-cell activity, as a result of CDR mutation and manipulation ofthe CDR and other sequences in the variable region to obtain a superiortherapeutic agent for the treatment of B-cell disorders, includingB-cell lymphomas and leukemias and autoimmune diseases. Modification tothe binding specificity, affinity or avidity of an antibody is known anddescribed in WO 98/44001, as affinity maturation, and this applicationsummarizes methods of modification and is incorporated in its entiretyby reference.

It may also be desirable to modify the antibodies of the presentinvention to improve effector function, e.g., so as to enhanceantigen-dependent cell-mediated cytotoxicity (ADCC) and/or complementdependent cytotoxicity (CDC) of the antagonist. One or more amino acidsubstitutions or the introduction of cysteine in the Fc region may bemade, thereby improving internalization capability and/or increasedcomplement-mediated cell killing and ADCC. See Caron et al., J. Ex. Med.176:1191-1195 (1991) and Shopes, Brit. J. Immunol. 148:2918-2022 (1992),incorporated herein by reference in their entirety. An antibody fusionprotein may be prepared that has dual Fc regions with both enhancedcomplement lysis and ADCC capabilites.

The present invention is also directed to DNA sequences comprising anucleic acid encoding a MAb or fragment thereof selected from the groupconsisting

(a) an anti-CD19 MAb or fragment thereof as described herein,

(b) an antibody fusion protein or fragment thereof comprising at leasttwo of the anti-CD19 MAbs or fragments thereof,

(c) an antibody fusion protein or fragment thereof comprising at leastone first MAb or fragment thereof comprising the anti-CD 19 MAb orfragment thereof as described herein and at least one second MAb orfragment thereof, other than the antiCD19 MAb or fragment thereof, and

(d) an antibody fusion protein or fragment thereof comprising at leastone first MAb or fragment thereof comprising the anti-CD19 MAb orfragment thereof and at least one second MAb or fragment thereof,wherein the second MAb is a MAb reactive with CD4, CD5, CD8, CD14, CD15,CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46,CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC1, 1a, HM1.24, HLA-DR,tenescin, ED-B fibronectin, IL-6, VEGF, PIGF, TRAIL-R1 (DR4) andTRAIL-R2 (DR5) or a combination thereof.

Also encompassed by the present invention are expression vectorscomprising the DNA sequences. These vectors contain the light and heavychain constant regions and the hinge region of the human immunoglobulin,in the case of vectors far use in preparing the humanized, chimeric andhuman anti-CD19 MAbs or antibody fusion proteins thereof or fragmentsthereof. These vectors additionally contain, where required, promotersthat express the MAbs in the selected host cell, immunaglobulinenhancers and signal or leader sequences. Vectors that are particularlyuseful in the present invention are pdHL2 or GS, particularly when usedto express a chimeric, humanized or human antibodies, such as IgGs,where the vector codes far the heavy and light chain constant regionsand hinge region of IgG1. More preferably, the light and heavy chainconstant regions and hinge region are from a human ED myelomaimmunoglobulin, where optionally at least one of the amino acid in theallotype positions is changed to that found in a different IgG1allotype, and wherein optionally amino acid 253 of the heavy chain of EUbased on the EU number system may be replaced with alanine. See Edelmanet al., Proc. Natl. Acad. Sci USA 63: 78-85 (1969), incorporated hereinin its entirety by reference.

Host cells containing the DNA sequences encoding the anti-CD 19 MAbs orfragments thereof or antibody fusion proteins or fragments thereof ofthe present invention or host cells containing the vectors that containthese DNA sequences are encompassed by the present invention.Particularly useful hast cells are mammalian cells, mare specificallylymphocytic cells, such as myeloma cells, discussed in more detailbelow.

Also encompassed by the present invention is the method of expressingthe anti-CD 19 MAb or fragment thereof or antibody fusion protein orfragment thereof comprising: (a) transfecting a mammalian cell with aDNA sequence of encoding the anti-CD19MAbs or fragments thereof orantibody fusion proteins or fragments thereof, and (b) culturing thecell transfected with the DNA sequence that secretes the anti-CD19 orfragment thereof or antibody fusion protein or fragment thereof. Knowntechniques may be used that include a selection marker on the vector sothat host cells that express the MAbs and the marker can be easilyselected.

The present invention particularly encompasses B-lymphoma cell andleukemia cell targeting diagnostic or therapeutic conjugates comprisingan antibody component comprising an anti-CD19 MAb or fragment thereof oran antibody fusion protein or fragment thereof of the present inventionthat binds to the B-lymphoma or leukemia cell that is bound to at leastone diagnostic or at least one therapeutic agent.

The diagnostic conjugate comprises the antibody component comprising ananti-CD19 MAb or fragment thereof or an antibody fusion protein orfragment thereof, wherein the diagnostic agent comprises at least onephotoactive diagnostic agent, and more preferably wherein the label is aradioactive label with an energy between 60 and 4,000 keV or anon-radioactive label. The radioactive label is preferably a gamma-,beta-, and positron-emitting isotope and is selected from the groupconsisting of ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁸⁶Y, 186Re, ¹⁸⁸Re, ⁶²Cu, ⁶⁴Cu,¹¹¹In, ⁶⁷G, ⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹⁸F, ¹¹C, ¹³N, ¹⁵0, ⁷⁶Br andcombinations thereof.

The diagnostic conjugate of the present invention also utilizes adiagnostic agent, such as a contrast agent, for example, such asmanganese, iron or gadolinium, and including an ultrasound-enhancingagent. In one embodiment, the ultrasound-enhancing agent is a liposomethat comprises a chimerized or humanized anti-CD19 antibody or fragmentthereof. Also preferred, the ultrasound enhancing agent is a liposomethat is gas filled. Similarly, a bispecific antibody can be conjugatedto a contrast agent. For example, the bispecific antibody may comprisemore than one image enhancing agent for use in ultrasound imaging. Theultrasound enhancing agent can be a liposome, and preferably, theliposome comprises a bivalent DTP A peptide covalently attached to theoutside surface of the liposome. Also preferred, the liposome is gasfilled.

The therapeutic conjugate of the present invention comprises an antibodycomponent comprising an antibody fusion protein or fragment thereof,wherein each of said MAbs or fragments thereof are bound to at least onetherapeutic agent. The therapeutic conjugate of preferably is selectedfrom the group consisting of a radioactive label, an immunomodulator, ahormone, an enzyme, an oligonucleotide, a photoactive therapeutic agent,a cytotoxic agent, which may be a drug or a toxin, and a combinationthereof. The drugs useful in the present invention are those drugs thatpossess the pharmaceutical property selected from the group consistingof antimitotic, alkylating, antimetabolite, antibiotic, alkaloid,antiangiogenic, apoptotic agents and combinations thereof, as well asantisense oligonucleotides and RNA molecules, such as short doublestranded RNA molecules that activate the RNA interference pathway. Morespecifically, these drugs are selected from the group consisting ofnitrogen mustards, ethylenimine derivatives, alkyl sulfonates,nitrosoureas, triazenes, folic acid analogs, COX-2 inhibitors,pyrimidine analogs, purine analogs, antibiotics, enzymes,epipodophyllotoxins, platinum coordination complexes, vinca alkaloids,substituted ureas, methyl hydrazine derivatives, adrenocorticalsuppressants, thalidomide and its derivatives, antagonists, endostatin,taxols, camptothecins, anthracyclines, taxanes, and their analogs, and acombination thereof. The toxins encompassed by the present invention areselected from the group consisting of ricin, abrin, alpha toxin,saporin, onconase, i.e., ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

Other therapeutic agents suitable for use in the present inventioninclude anti-angiogenic agents (or angiogenesis inhibitors). Theseagents are suitable for use in combination therapy or for conjugatingantibodies to, for example, angiostatin, endostatin, vasculostatin,canstatin and maspin, as well as the use of antibodies againstangiogenesis factors, such as vascular endothelium growth factor (VEGF),placental growth factor (PIGF), ED-B fibronectin, and against othervascular growth factors. Single and double stranded oligonucleotides area new class of therapeutic agents, and include, for example, antisenseoligonucleotides, such as antisense bcl-2, and molecules, such as doublestranded RNA molecules, that activate the RNA interference pathway andcause highly specific inhibition of gene expression, such as inhibitionof bcl-2. Inhibition of bcl-2 (and related bcl family molecules) in acell inhibits the anti-apoptotic activity of bcl-2 and promotesapoptosis of the cell. See Zangemeister-Wittke, Ann N Y Acad Sci.1002:90-4 (2003).

Useful therapeutic conjugates of the present invention areimmunomodulators selected from the group consisting of a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof. Specifically useful arelymphotoxins, such as tumor necrosis factor (TNF), hematopoieticfactors, such as interleukin (IL), colony stimulating factor, such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF)), interferon, such asinterferons-, alpha-, beta- or gamma-, and stem cell growth factor, suchas designated “S1 factor”. More specifically, immunomodulator, such asIL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, interferon-, TNF- ora combination thereof are useful in the present invention.

Particularly useful therapeutic conjugates are one or more radioactivelabels that have an energy between 60 and 700 ke V. Such radioactivelabels are selected from the group consisting of ²²⁵Ac, ⁶⁷Ga ⁹⁰Y, ¹¹¹In,¹³¹I, ¹²⁵I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ³²P, ⁶⁴Cu, ⁶⁷Cu, ²¹²Bi, ²¹³Bi, ²¹¹Atand combinations thereof. Other useful therapeutic conjugates arephotoactive therapeutic agent, such as a chromogen or dye.

The present invention particularly encompasses methods of treating aB-cell disease in a subject, such as a mammal, including humans,domestic or companion pets, such as dogs and cats. B cell diseases thatcan be treated by the methods of the invention include any disease whichinvolves unwanted or undesirable B cell growth or activity, and includesmalignancies such as lymphoma or leukemia cell disease or an autoimmunedisease. The methods involve administering to the subject atherapeutically effective amount of an anti-CD19 MAb or a fragmentthereof of the present invention, formulated in a pharmaceuticallyacceptable vehicle. This therapy utilizes a “naked antibody” that doesnot have a therapeutic agent bound to it. The administration of the“naked anti-CD19 antibody” can be supplemented by administering to thesubject concurrently or sequentially a therapeutically effective amountof another “naked antibody” that binds to or is reactive with anotherantigen on the surface of the target cell or that has other functions,such as effector functions in the Fc portion of the MAb, that istherapeutic and which is discussed herein. Preferred additional MAbs areat least one humanized, chimeric, human or murine (in the case ofnon-human animals) MAb selected from the group consisting of a MAbreactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23,CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD74,CD80, CD126, CD138, B7, HM1.24, HLA-DR, an angiogenesis factor,tenascin, VEGF, PIGF, ED-B fibronectin, an oncogene, an oncogeneproduct, NCA 66a-d, necrosis antigens, Ii, IL-2, MUC-1, TO1, TAC, IL-6,TRAIL-R1 (DR4) and TRAIL-R2 (DR5) formulated in a pharmaceuticallyacceptable vehicle.

Both the naked anti-CD19 therapy alone or in combination with othernaked MAbs as discussed above can be further supplemented with theadministration, either concurrently or sequentially, of atherapeutically effective amount of at least one therapeutic agent,formulated in a pharmaceutically acceptable vehicle. As discussed hereinthe therapeutic agent may comprise a cytotoxic agent, a radioactivelabel, an immunomodulator, a hormone, an oligonucleotide (such as anantisense or RNAi oligonucleotide), an enzyme, a photoactive therapeuticagent or a combination thereof, formulated in a pharmaceuticallyacceptable vehicle.

In another therapeutic method, both the naked anti-CD 19 therapy aloneor in combination with other naked MAbs, as discussed above, can befurther supplemented with the administration, either concurrently orsequentially, of a therapeutically effective amount of at least onetherapeutic conjugate, described herein and formulated in apharmaceutically acceptable vehicle. The antibody component of thetherapeutic conjugate comprises at least one humanized, chimeric, humanor murine (for non-human subjects) MAb selected from the groupconsisting of a MAb reactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20,CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54,CD74, CD80, CD126, CD138, B7, HM1.24, HLA-DR, an angiogenesis factor,tenascin, VEGF, PIGF, ED-B fibronectin, an oncogene, an oncogeneproduct, NCA 66a-d, necrosis antigens, Ii, IL-2, T101, TAC, IL-6, MUC-1,TRAIL-R1 (DR4) and TRAIL-R2 (DR5), formulated in a pharmaceuticallyacceptable vehicle. As discussed herein the therapeutic agent maycomprise a cytotoxic agent, a radioactive label, an immunomodulator, ahormone, a photoactive therapeutic agent or a combination thereof,formulated in a pharmaceutically acceptable vehicle.

As described herein the present invention particurlarly encompasses amethod of treating a B-cell lymphoma or leukemia or an autoimmunedisease in a subject comprising administering to a subject atherapeutically effective amount of an antibody fusion protein orfragment thereof comprising at least two anti-CD 19 MAbs or fragmentsthereof of the present invention or comprising at least one anti-CD19MAb or fragment thereof of the present invention and at least oneadditional MAb, preferably selected from the group consisting of MAbsreactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23,CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80,CD126, CD138, B7, HM1.24, HLA-DR, tenascin, VEGF, PIGF, ED-Bfibronectin, MUC-1, an oncogene, an oncogene product, NCA 66a-d,necrosis antigens, Ii, IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4) andTRAIL-R2 (DR5) formulated in a pharmaceutically acceptable vehicle.

This therapeutic method can further be supplemented with theadministration to the subject concurrently or sequentially of atherapeutically effective amount of at least one therapeutic agent,formulated in a pharmaceutically acceptable vehicle, wherein thetherapeutic agent is preferably a cytotoxic agent, a radioactive label,an immunomodulator, a hormone, a photoactive therapeutic agent or acombination thereof, formulated in a pharmaceutically acceptablevehicle.

Further, the antibody fusion proteins can be administered to a subjectconcurrently or sequentially a therapeutically effective amount of atherapeutic conjugate comprising at least one MAb bound to at least onetherapeutic agent, wherein said MAb component of the conjugatepreferably comprises at least one humanized, chimeric, human or murine(for non-human subjects) MAb selected from the group consisting of a MAbreactive with CD4, CD5, CD8, CD 14, CD15, CD19, CD20, CD21, CD22, CD23,CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80,CD126, CD138, B7, MUC1, Ia, HM1.24, HLA-DR, tenascin, VEGF, PIGF, ED-Bfibronectin, an oncogene, an oncogene product, NCA 66a-d, necrosisantigens, Ii, IL-2, IL-6, T101, TAC, IL-6, TRAIL-R1 (DR4) and TRAIL-R2(DR5) formulated in a pharmaceutically acceptable vehicle. The antibodyfusion protein itself can be conjugated to a therapeutic agent and thusprovides a vehicle to attach more than one therapeutic agent to anantibody component and these therapeutic agents can be a combination ofdifferent recited agents or combinations of the same agents, such as twodifferent therapeutic radioactive labels.

Also encompassed by the present invention is a method of diagnosing aB-cell lymphoma or leukemia in a subject comprising administering to thesubject, such as a mammal, including humans and domestic and companionpets, such as dogs, cats, rabbits, guinea pigs, a diagnostic conjugatecomprising an anti-CD19 MAb or fragment thereof or an antibody fusionprotein or fragment thereof of the present invention that binds to thelymphoma or leukemia cell, wherein the anti-CD 19 MAb or fragmentthereof or antibody fusion protein or fragment thereof is bound to atleast one diagnostic agent, formulated in a pharmaceutically acceptablevehicle. The useful diagnostic agents are described herein.

2. Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of thepresent invention.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab)₂, F(ab)₂,Fab, Fab, Fv, sFv and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-CD19 monoclonal antibody fragment bindswith an epitope of CD19. The term “antibody fragment” also includes anysynthetic or genetically engineered protein that acts like an antibodyby binding to a specific antigen to form a complex. For example,antibody fragments include isolated fragments consisting of the variableregions, such as the “Fv” fragments consisting of the variable regionsof the heavy and light chains, recombinant single chain polypeptidemolecules in which light and heavy variable regions are connected by apeptide linker (“scFv proteins”), and minimal recognition unitsconsisting of the amino acid residues that mimic the hypervariableregion.

A naked antibody is generally an entire antibody which is not conjugatedto a therapeutic agent. This is so because the Fc portion of theantibody molecule provides effector functions, such as complementfixation and ADCC (antibody dependent cell cytotoxicity), which setmechanisms into action that may result in cell lysis. However, it ispossible that the Fc portion is not required for therapeutic function,with other mechanisms, such as apoptosis, coming into play. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric, humanized or humanantibodies.

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule is derived from those of ahuman antibody. For veterinary applications, the constant domains of thechimeric antibody may be derived from that of other species, such as acat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, is transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains. The constant domains of the antibodymolecule is derived from those of a human antibody.

A human antibody is an antibody obtained from transgenic mice that havebeen “engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain locus are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incoporated intheir entirety by reference.

A therapeutic agent is a molecule or atom which is administeredseparately, concurrently or sequentially with an antibody moiety orconjugated to an antibody moiety, i.e., antibody or antibody fragment,or a sub fragment, and is useful in the treatment of a disease. Examplesof therapeutic agents include antibodies, antibody fragments, drugs,toxins, enzymes, oligonucleotides, antisense and RNAi oligonucleotides,nucleases, hormones, immunomodulators, chelators, boron compounds,photoactive agents or dyes and radioisotopes.

A diagnostic agent is a molecule or atom which is administeredconjugated to an antibody moiety, i.e., antibody or antibody fragment,or subfragment, and is useful in diagnosing a disease by locating thecells containing the antigen. Useful diagnostic agents include, but arenot limited to, radioisotopes, dyes (such as with thebiotin-streptavidin complex), contrast agents, ultrasound-enhancingagents, optical-enhancing agents, fluorescent compounds or molecules andenhancing agents (e.g. paramagnetic ions) for magnetic resonance imaging(MRI). U.S. Pat. No. 6,331,175 describes MRI technique and thepreparation of antibodies conjugated to a MRI enhancing agent and isincorporated in its entirety by reference. Preferably, the diagnosticagents are selected from the group consisting of radioisotopes,enhancing agents for use in magnetic resonance imaging, ultrasound, andfluorescent compunds. In order to load an antibody component withradioactive metals or paramagnetic ions, it may be necessary to react itwith a reagent having a long tail to which are attached a multiplicityof chelating groups for binding the ions. Such a tail can be a polymersuch as a polylysine, polysaccharide, or other derivatized orderivatizable chain having pendant groups to which can be boundchelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crownethers, bis-thiosemicarbazones, polyoximes, and like groups known to beuseful for this purpose. Chelates are coupled to the peptide antigensusing standard chemistries. The chelate is normally linked to theantibody by a group which enables formation of a bond to the moleculewith minimal loss of immunoreactivity and minimal aggregation and/orinternal cross-linking. Other, more unusual, methods and reagents forconjugating chelates to antibodies are disclosed in U.S. Pat. No.4,824,659 to Hawthorne, entitled “Antibody Conjugates”, issued Apr. 25,1989, the disclosure of which is incorporated herein in its entirety byreference. Particularly useful metal-chelate combinations include2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used withdiagnostic isotopes in the general energy range of 60 to 4,000 keV, suchas ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁶²Cu ⁶²Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc,^(94m)Tc, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, for radio-imaging. The same chelates,when complexed with non-radioactive metals, such as manganese, iron andgadolinium are useful for MRI, when used along with the antibodies ofthe invention. Macrocyclic chelates such as NOTA, DOTA, and TETA are ofuse with a variety of metals and radiometals, most particularly withradionuclides of gallium, yttrium and copper, respectively. Suchmetal-chelate complexes can be made very stable by tailoring the ringsize to the metal of interest. Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides,such as ²²³Ra for RAIT are encompassed by the invention.

An immunoconiugate is a conjugate of an antibody component with atherapeutic or diagnostic agent. The diagnostic agent can comprise aradioactive or non-radioactive label, a contrast agent (such as formagnetic resonance imaging, computed tomography or ultrasound), and theradioactive label can be a gamma-, beta-, alpha-, Auger electron-, orpositron-emitting isotope.

An expression vector is a DNA molecules comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells, as well as an transgenicanimal, that have been genetically engineered to contain the clonedgene(s) in the chromosome or genome of the host cell or cells of thehost cells. Suitable mammalian host cells include myeloma cells, such asSP2/0 cells, and NS0 cells, as well as Chinese Hamster Ovary (CH0)cells, hybridoma cell lines and other mammalian host cell useful forexpressing antibodies. Also particularly useful to express MAbs andother fusion proteins, is a human cell line, PER.C6 disclosed in WO0063403 A2, which produces 2 to 200-fold more recombinant protein ascompared to conventional mammalian cell lines, such as CHO, COS, Vero,Hela, BHK and SP2-cell lines. Special transgenic animals with a modifiedimmune system are particularly useful for making fully human antibodies.

As used herein, the term antibody fusion protein is a recombinantlyproduced antigen-binding molecule in which two or more of the same ordifferent single-chain antibody or antibody fragment segments with thesame or different specificities are linked. Valency of the fusionprotein indicates how many binding arms or sites the fusion protein hasto a single antigen or epitope; i.e., monovalent, bivalent, trivalent ormutlivalent. The multivalency of the antibody fusion protein means thatit can take advantage of multiple interactions in binding to an antigen,thus increasing the avidity of binding to the antigen. Specificityindicates how many antigens or epitopes an antibody fusion protein isable to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Monospecific, multivalent fusionproteins have more than one binding site for an epitope but only bindswith one epitope, for example a diabody with two binding site reactivewith the same antigen. The fusion protein may comprise a single antibodycomponent, a multivalent or multispecific combination of differentantibody components or multiple copies of the same antibody component.The fusion protein may additionally comprise an antibody or an antibodyfragment and a therapeutic agent. Examples of therapeutic agentssuitable for such fusion proteins include immunomodulators(“antibody-immunomodulator fusion protein”) and toxins (“antibody-toxinfusion protein”). One preferred toxin comprises a ribonuclease (RNase),preferably a recombinant RNase.

A multispecific antibody is an antibody that can bind simultaneously toat least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. One specificity would be for aB-cell, T-cell, myeloid-, plasma-, and mast-cell antigen or epitope.Another specificity could be to a different antigen on the same celltype, such as CD20, CDI9, CD20, CD21, CD23, CD46, CD80, HLA-DR, CD74,and CD22 on B-cells. Multispecific, multivalent antibodies areconstructs that have more than one binding site, and the binding sitesare of different specificity. For example, a diabody, where one bindingsite reacts with one antigen and the other with another antigen.

A bispecific antibody is an antibody that can bind simultaneously to twotargets which are of different structure. Bispecific antibodies (bsAb)and bispecific antibody fragments (bsFab) have at least one arm thatspecifically binds to, for example, a B-cell, T-cell, myeloid-, plasma-,and mast-cell antigen or epitope and at least one other arm thatspecifically binds to a targetable conjugate that bears a therapeutic ordiagnostic agent. A variety of bispecific fusion proteins can beproduced using molecular engineering. In one form, the bispecific fusionprotein is monovalent, consisting of, for example, a scFv with a singlebinding site for one antigen and a Fab fragment with a single bindingsite for a second antigen. In another form, the bispecific fusionprotein is divalent, consisting of, for example, an IgG with a bindingsite for one antigen and two scFv with two binding sites for a secondantigen.

Caninized or felinized antibodies are recombinant proteins in whichrodent (or another species) complementarity determining regions of amonoclonal antibody have been transferred from heavy and light variablechains of rodent (or another species) immunoglobulin into a dog or cat,respectively, immunoglobulin variable domain.

Domestic animals include large animals such as horses, cattle, sheep,goats, llamas, alpacas, and pigs, as well as companion animals. In apreferred embodiment, the domestic animal is a horse.

Companion animals include animals kept as pets. These are primarily dogsand cats, although small rodents, such as guinea pigs, hamsters, rats,and ferrets, are also included, as are subhuman primates such asmonkeys. In a preferred embodiment the companion animal is a dog or acat.

3. Preparation of Monoclonal Antibodies Including Chimeric, Humanizedand Human Antibodies

Monoclonal antibodies (MAbs) are a homogeneous population of antibodiesto a particular antigen and the antibody comprises only one type ofantigen binding site and binds to only one epitope on an antigenicdeterminant. Rodent monoclonal antibodies to specific antigens may beobtained by methods known to those skilled in the art. See, for example,Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al. (eds.),CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley &Sons 1991) [hereinafter “Coligan”]. Briefly, monoclonal antibodies canbe obtained by injecting mice with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B-lymphocytes, fusing theB-Iymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to theantigen, culturing the clones that produce antibodies to the antigen,and isolating the antibodies from the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are wen known to those skilled in the art. For example,humanized monoclonal antibodies are produced by transferring mousecomplementary determining regions from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, and then,substituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions.

General techniques for cloning murine immunoglobulin variable domainsare described, for example, by the publication of Orlandi et al., Proc.Natfl Acad. Sci. USA 86: 3833 (1989), which is incorporated by referencein its entirety. Techniques for constructing chimeric antibodies arewell known to those of skill in the art. As an example, Leung et at.,Hybridoma 13:469 (1994), describe how they produced an LL2 chimera bycombining DNA sequences encoding the V and V_(H) domains of LL2monoclonal antibody, an anti-CD22 antibody, with respective human andIgG₁ constant region domains. This publication also provides thenucleotide sequences of the LL2 light and heavy chain variable regions,V and V_(H), respectively. Techniques for producing humanized MAbs aredescribed, for example, by Jones et al., Nature 321: 522 (1986),Riechmann et al., Nature 332: 323 (1988), Verhoeyen et al., Science 239:1534 (1988), Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992),Sandhu, Crit. Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun.150: 2844 (1993), each of which is hereby incorporated by reference.

A chimeric antibody is a recombinant protein that contains the variabledomains including the CDRs derived from one species of animal, such as arodent antibody, while the remainder of the antibody molecule; i.e., theconstant domains, is derived from a human antibody. Accordingly, achimeric monoclonal antibody can also be humanized by replacing thesequences of the murine FR in the variable domains of the chimeric MAbwith one or more different human FR. Specifically, mouse CDRs aretransferred from heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. As simply transferring mouse CDRs into human FRs often resultsin a reduction or even loss of antibody affinity, additionalmodification might be required in order to restore the original affinityof the murine antibody. This can be accomplished by the replacement ofone or more some human residues in the FR regions with their murinecounterparts to obtain an anatibody that possesses good binding affinityto its epitope. See, for example, Tempest et al., Biotechnology 9:266(1991) and Verhoeyen et al., Science 239: 1534 (1988). Further, theaffinity of humanized, chimeric and human MAbs to a specific epitope canbe increased by mutagenesis of the CDRs, so that a lower dose ofantibody may be as effective as a higher dose of a lower affinity MAbprior to mutagenesis. See for example, WO0029584A1.

Another method for producing the antibodies of the present invention isby production in the milk of transgenic livestock. See, e.g., Colman,A., Biochem. Soc. Symp., 63: 141-147, 1998; U.S. Pat. No. 5,827,690,both of which are incorporated in their entirety by reference. Two DNAconstructs are prepared which contain, respectively, DNA segmentsencoding paired immunoglobulin heavy and light chains. The DNA segmentsare cloned into expression vectors which contain a promoter sequencethat is preferentially expressed in mammary epithelial cells. Examplesinclude, but are not limited to, promoters from rabbit, cow and sheepcasein genes, the cow-lactoglobulin gene, the sheep-lactoglobulin geneand the mouse whey acid protein gene. Preferably, the inserted fragmentis flanked on its 3′ side by cognate genomic sequences from amammary-specific gene. This provides a polyadenylation site andtranscript-stabilizing sequences. The expression cassettes arecoinjected into the pronuclei of fertilized, mammalian eggs, which arethen implanted into the uterus of a recipient female and allowed togestate. After birth, the progeny are screened for the presence of bothtransgenes by Southern analysis. In order for the antibody to bepresent, both heavy and light chain genes must be expressed concurrentlyin the same cell. Milk from transgenic females is analyzed for thepresence and functionality of the antibody or antibody fragment usingstandard immunological methods known in the art. The antibody can bepurified from the milk using standard methods known in the art.

A fully human antibody of the present invention, i.e., human anti-CD19MAbs or other human antibodies, such as anti-CD22, anti-CD19, anti-CD23,anti-CD20 or anti-CD21 MAbs for combination therapy with humanized,chimeric or human anti-CD19 antibodies, can be obtained from atransgenic non-human animal. See, e.g., Mendez et al., Nature Genetics,15: 146-156 (1997); U.S. Pat. No. 5,633,425, which are incorporated intheir entirety by reference. For example, a human antibody can berecovered from a transgenic mouse possessing human immunoglobulin loci.The mouse humoral immune system is humanized by inactivating theendogenous immunoglobulin genes and introducing human immunoglobulinloci. The human immunoglobulin loci are exceedingly complex and comprisea large number of discrete segments which together occupy almost 0.2% ofthe human genome. To ensure that transgenic mice are capable ofproducing adequate repertoires of antibodies, large portions of humanheavy- and light-chain loci must be introduced into the mouse genome.This is accomplished in a stepwise process beginning with the formationof yeast artificial chromosomes (YACs) containing either human heavy orlight-chain immunoglobulin loci in germline configuration. Since eachinsert is approximately 1 Mb in size, YAC construction requireshomologous recombination of overlapping fragments of the immunoglobulinloci. The two YACs, one containing the heavy-chain loci and onecontaining the light-chain loci, are introduced separately into mice viafusion of YAC-containing yeast spheroblasts with mouse embryonic stemcells. Embryonic stem cell clones are then microinjected into mouseblastocysts. Resulting chimeric males are screened for their ability totransmit the Y AC through their germline and are bred with micedeficient in murine antibody production. Breeding the two transgenicstrains, one containing the human heavy-chain loci and the othercontaining the human light-chain loci, creates progeny which producehuman antibodies in response to immunization.

Further recent methods for producing bispecific MAbs include engineeredrecombinant MAbs which have additional cysteine residues so that theycrosslink more strongly than the more common immunoglobulin isotypes.See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225, 1997.Another approach is to engineer recombinant fusion proteins linking twoor more different single-chain antibody or antibody fragment segmentswith the needed dual specificities. See, e.g., Coloma et al., NatureBiotech. 15:159-163, 1997. A variety of bispecific fusion proteins canbe produced using molecular engineering. See, for example, Alt et al.,FEBS Lett. 454:90-4 (1999), which is incorporated herein by reference inits entirety. In one form, the bispecific fusion protein is monovalent,consisting of, for example, a scFv with a single binding site for oneantigen and a Fab fragment with a single binding site for a secondantigen. In another form, the bispecific fusion protein is divalent,consisting of, for example, an IgG with two binding sites for oneantigen and two scFv with two binding sites for a second antigen.

Bispecific fusion proteins linking two or more different single-chainantibodies or antibody fragments are produced in similar manner.Recombinant methods can be used to produce a variety of fusion proteins.For example a fusion protein comprising a Fab fragment derived from ahumanized monoclonal anti-CD19 antibody and a scFv derived from a murineanti-diDTPA can be produced. A flexible linker, such as GGGS connectsthe scFv to the constant region of the heavy chain of the anti-CD19antibody. Alternatively, the scFv can be connected to the constantregion of the light chain of another humanized antibody. Appropriatelinker sequences necessary for the in-frame connection of the heavychain Fd to the scFv are introduced into the VL and VK domains throughPCR reactions. The DNA fragment encoding the scFv is then ligated into astaging vector containing a DNA sequence encoding the CH1 domain. Theresulting scFv-CH1 construct is excised and ligated into a vectorcontaining a DNA sequence encoding the VH region of an anti-CD19antibody. The resulting vector can be used to transfect an appropriatehost cell, such as a mammalian cell for the expression of the bispecificfusion protein.

4. Production of Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab′)₂, Fab′, Fab, Fv, sFv and the like. Otherantibody fragments include, but are not limited to: the F(ab)′₂fragments which can be produced by pepsin digestion of the antibodymolecule and the Fab′ fragments, which can be generated by reducingdisulfide bridges of the F(ab)′2 fragments. Alternatively, Fab′expression expression libraries can be constructed (Huse et al., 1989,Science, 246:1274-1281) to allow rapid and easy identification ofmonoclonal Fab′ fragments with the desired specificity. The presentinvention encompasses antibodies and antibody fragments.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). A scFvmolecule is denoted as either VL-L-VH if the VL domain is the N-terminalpart of the scFv molecule, or as VH-L-VL if the VH domain is theN-terminal part of the scFv molecule. Methods for making scFv moleculesand designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, “SingleChain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker,“Single Chain Antibody Variable Regions,” TIBTECH, Vol 9: 132-137(1991). These references are incorporated herein by reference.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull-length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment denotedF(ab)₂. This fragment can be further cleaved using a thiol reducingagent, and optionally a blocking group for the sulfhydryl groupsresulting from cleavage of disulfide linkages, to produce 3.5S Fabmonovalent fragments. Alternatively, an enzymatic cleavage using papainproduces two monovalent Fab fragments and an Fc fragment directly. Thesemethods are described, for example, by Goldenberg, U.S. Pat. Nos.4,036,945 and 4,331,647 and references contained therein, which patentsare incorporated herein in their entireties by reference. Also, seeNisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem.J. 73: 119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). A CDR is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. See, for example, Larrick et al., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoctonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

5. Multispecific and Multivalent Antibodies

The anti-CD19 antibodies, as well as other antibodies with differentspecificities for use in combination therapy, described herein, can alsobe made as multispecific antibodies (comprising at least one bindingsite to a CD19 epitope or antigen and at least one binding site toanother epitope on CD19 or another antigen) and multivalent antibodies(comprising mutliple binding sites to the same epitope or antigen).

The present invention provides a bispecific antibody or antibodyfragment having at least a binding region that specifically binds atargeted cell marker and at least one other binding region thatspecifically binds a targetable conjugate. The targetable conjugatecomprises a carrier portion which comprises or bears at least oneepitope recognized by at least one binding region of the bispecificantibody or antibody fragment.

A variety of recombinant methods can be used to produce bispecificantibodies and antibody fragments as described above.

An anti-CD 19 multivalent antibody is also contemplated in the presentinvention. This multivalent target binding protein is constructed byassociation of a first and a second polypeptide. The first polypeptidecomprises a first single chain Fv molecule covalently linked to a firstimmunoglobulin-like domain which preferably is an immunoglobulin lightchain variable region domain. The second polypeptide comprises a secondsingle chain Fv molecule covalently linked to a secondimmunoglobulin-like domain which preferably is an immunoglobulin heavychain variable region domain. Each of the first and second single chainFv molecules forms a target binding site, and the first and secondimmunoglobulin-like domains associate to form a third target bindingsite.

A single chain Fv molecule with the VL-L-VH configuration, wherein L isa linker, may associate with another single chain Fv molecule with theVH-L-VL configuration to form a bivalent dimer. In this case, the VLdomain of the first scFv and the VH domain of the second scFv moleculeassociate to form one target binding site, while the VH domain of thefirst scFv and the VL domain of the second scFv associate to form theother target binding site.

Another embodiment of the present invention is a CD19 bispecific,trivalent targeting protein comprising two heterologous polypeptidechains associated noncovalently to form three binding sites, two ofwhich have affinity for one target and a third which has affinity for ahapten that can be made and attached to a carrier for a diagnosticand/or therapeutic agent. Preferably, the binding protein has two CD19binding sites and one CD22 binding site. The bispecific, trivalenttargeting agents have two different scFvs, one scFv contains two V_(H)domains from one antibody connected by a short linker to the V_(L)domain of another antibody and the second scFv contains two V_(L)domains from the first antibody connected by a short linker to the V_(H)domain of the other antibody. The methods for generating multivalent,multispecific agents from V_(H) and V_(L) domains provide thatindividual chains synthesized from a DNA plasmid in a host organism arecomposed entirely of V_(H) domains (the V_(H)-chain) or entirely ofV_(L) domains (the V_(L)-chain) in such a way that any agent ofmultivalency and multispecificity can be produced by non-covalentassociation of one V_(H)-chain with one V_(L)-chain. For example,forming a trivalent, trispecific agent, the V_(H)-chain will consist ofthe amino acid sequences of three V_(H) domains, each from an antibodyof different specificity, joined by peptide linkers of variable lengths,and the V_(L)-chain will consist of complementary V_(L) domains, joinedby peptide linkers similar to those used for the V_(H)-chain. Since theV_(H) and V_(L) domains of antibodies associate in an anti-parallelfashion, the preferred method in this invention has the V_(L) domains inthe V_(L)-chain arranged in the reverse order of the V_(H) domains inthe V_(H)-chain.

6. Diabodies, Triabodies and Tetrabodies

The anti-CD19 antibodies of the present invention can also be used toprepare functional bispecific single-chain antibodies (bscAb), alsocalled diabodies, and can be produced in mammalian cells usingrecombinant methods. See, e.g., Mack et al., Proc. Natl. Acad. Sci., 92:7021-7025, 1995, incorporated. For example, bscAb are produced byjoining two single-chain Fv fragments via a glycine-serine linker usingrecombinant methods. The V light-chain (V_(L)) and V heavy-chain (V_(H))domains of two antibodies of interest are isolated using standard PCRmethods. The V_(L) and V_(H) eDNA's obtained from each hybridoma arethen joined to form a single-chain fragment in a two-step fusion PCR.The first PCR step introduces the (Gly₄-Ser₁)₃ linker, and the secondstep joins the V_(L) and V_(H) amplicons. Each single chain molecule isthen cloned into a bacterial expression vector. Following amplification,one of the single-chain molecules is excised and sub-cloned into theother vector, containing the second single-chain molecule of interest.The resulting bscAb fragment is subcloned into an eukaryotic expressionvector. Functional protein expression can be obtained by transfectingthe vector into chinese hamster ovary cells. Bispecific fusion proteinsare prepared in a similar manner. Bispecific single-chain antibodies andbispecific fusion proteins are included within the scope of the presentinvention.

For example, a humanized, chimeric or human anti-CD19 monoclonalantibody can be used to produce antigen specific diabodies, triabodies,and tetrabodies. The monospecific diabodies, triabodies, and tetrabodiesbind selectively to targeted antigens and as the number of binding siteson the molecule increases, the affinity for the target cell increasesand a longer residence time is observed at the desired location. Fordiabodies, the two chains comprising the V_(H) polypeptide of thehumanized CD19 MAb connected to the V_(K) polypeptide of the humanizedCD19 MAb by a five amino acid residue linker are utilized. Each chainforms one half of the humanized CD19 diabody. In the case of triabodies, the three chains comprising V_(H) polypeptide of the humanizedCD19 MAb connected to the V_(K) polypeptide of the humanized CD19 MAb byno linker are utilized. Each chain forms one third of the hCD19triabody.

The ultimate use of the bispecific diabodies described herein is forpretargeting CD 19 positive tumors for subsequent specific delivery ofdiagnostic or therapeutic agents. These diabodies bind selectively totargeted antigens allowing for increased affinity and a longer residencetime at the desired location. Moreover, non-antigen bound diabodies arecleared from the body quickly and exposure of normal tissues isminimized. Bispecific antibody point mutations for enhancing the rate ofclearance can be found in U.S. Provisional Application No. 60/361,037 toQu et al. , which is incorporated herein by reference in its entirety.Bispecific diabodies for affinity enhancement are disclosed in U.S.application Ser. No. 10/270,071 (Rossi et al.), Ser. No. 10/270,073(Rossi et al.) and Ser. No. 10/328,190 (Rossi et al.), which areincorporated herein by reference in their entirety. The diagnostic andtherapeutic agents can include isotopes, drugs, toxins, cytokines,hormones, enzymes, oligonucleotides, growth factors, conjugates,radionuclides, and metals. For example, gadolinium metal is used formagnetic resonance imaging (MRI). Examples of radio nuclides are ²²⁵Ac,¹⁸F, ⁶⁸Ga, ⁶⁷Ga, ⁹⁰Y, ⁸⁶Y, ¹¹¹In, ¹³¹I, ¹²⁵I, ¹²³I, ^(99m)Tc, ^(99m)Tc,¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ²¹²Bi, ²¹³Bi, ³²P, ¹¹C, ¹³N, ¹⁵O,⁷⁶Br, and ²¹¹At. Other radionuclides are also available as diagnosticand therapeutic agents, especially those in the energy range of 60 to4,000 keV.

More recently, a tetravalent tandem diabody (termed tandab) with dualspecificity has also been reported (Cochlovius et al., Cancer Research(2000) 60: 4336-4341). The bispecific tandab is a dimer of two identicalpolypeptides, each containing four variable domains of two differentantibodies (V_(H1), V_(L1), V_(H2), V_(L2)) linked in an orientation tofacilitate the formation of two potential binding sites for each of thetwo different specificities upon self-association.

7. Conjugated Multivalent and Multispecific Anti-CD19 Antibodies

In another embodiment of the instant invention is a conjugatedmultivalent anti-CD19 antibody. Compositions and methods formultivalent, multispecific agents are described in Rossi et al., U.S.Patent Application Ser. No.: 60/436,359, filed Dec. 24, 2002, and U.S.Patent Application Ser. No. 60/464,532, filed Apr. 23, 2003, which areincorporated herein by reference in its entirety.

Additional amino acid residues may be added to either the N- orC-terminus of the first or the second polypeptide. The additional aminoacid residues may comprise a peptide tag, a signal peptide, a cytokine,an enzyme (for example, a pro-drug activating enzyme), a hormone, apeptide toxin, such as pseudomonas extoxin, a peptide drug, a cytotoxicprotein or other functional proteins. As used herein, a functionalprotein is a protein which has a biological function.

In one embodiment, drugs, toxins, radioactive compounds, enzymes,hormones, oligonucleotides, cytotoxic proteins, chelates, cytokines andother functional agents may be conjugated to the multivalent targetbinding protein, preferably through covalent attachments to the sidechains of the amino acid residues of the multivalent target bindingprotein, for example amine, carboxyl, phenyl, thiol or hydroxyl groups.Various conventional linkers may be used for this purpose, for example,diisocyanates, diisothiocyanates, bis(hydroxysuccinimide) esters,carbodiimides, maleimide-hydroxysuccinimide esters, glutaraldehyde andthe like. Conjugation of agents to the multivalent protein preferablydoes not significantly affect the protein's binding specificity oraffinity to its target. As used herein, a functional agent is an agentwhich has a biological function. A preferred functional agent is acytotoxic agent.

As discussed above, enzymes are also useful therapeutic agents. Forexample, alkaline phosphatase for use in combination withphosphate-containing prodrugs (U.S. Pat. No. 4,975,278); arylsulfatasefor use in combination with sulfate-containing prodrugs (U.S. Pat. No.5,270,196); peptidases and proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidase (U.S. Pat. Nos. 5,660,829;5,587,161; 5,405,990) and cathepsins (including cathepsin B and L), foruse in combination with peptide-based prodrugs;D-alanylcarboxypeptidases for use in combination with D-aminoacid-modified prodrugs; carbohydrate-cleaving enzymes such asbeta-galactosidase and neuraminidase for use in combination withglycosylated prodrugs (U.S. Pat. Nos. 5,561,119; 5,646,298);beta.-Iactamase for use in combination with beta-Iactam-containingprodrugs; penicillin amidases, such as penicillin-V-amidase (U.S. Pat.No. 4,975,278) or penicillin-G-amidase, for use in combination withdrugs derivatized at their amino nitrogens with phenoxyacetamide orphenylacetamide groups; and cytosine deaminase (U.S. Pat. Nos.5,338,678; 5,545,548) for use in combination with 5-fluorocytosine-basedprodrugs (U.S. Pat. No. 4,975,278), are suitable therapeutic agents forthe present invention.

In still other embodiments, bispecific antibody-directed delivery oftherapeutics or prodrug polymers to in vivo targets can be combined withbispecific antibody delivery of radionuclides, such that combinationchemotherapy and radioimmunotherapy is achieved. Each therapy can beconjugated to the targetable conjugate and administered simultaneously,or the nuclide can be given as part of a first targetable conjugate andthe drug given in a later step as part of a second targetable conjugate.

In another embodiment, cytotoxic agents may be conjugated to a polymericcarrier, and the polymeric carrier may subsequently be conjugated to themultivalent target binding protein. For this method, see Ryser et aI.,Proc. Natl. Acad. Sci. USA, 75:3867-3870,1978, U.S. Pat. No. 4,699,784and U.S. Pat. No. 4,046,722, which are incorporated herein by reference.Conjugation preferably does not significantly affect the bindingspecificity or affinity of the multivalent binding protein.

8. Humanized, Chimeric and Human Antibodies Use for Treatment andDiagnosis

Humanized, chimeric and human monoclonal antibodies, i.e., anti-CD19MAbs and other MAbs described herein, in accordance with this invention,are suitable for use in therapeutic methods and diagnostic methods.Accordingly, the present invention contemplates the administration ofthe humanized, chimeric and human antibodies of the present inventionalone as a naked antibody or administered as a multimodal therapy,temporally according to a dosing regimen, but not conjugated to, atherapeutic agent. The efficacy of the naked anti-CD19 MAbs can beenhanced by supplementing naked antibodies with one or more other nakedantibodies, i.e., MAbs to specific antigens, such as CD4, CD5, CD8,CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40,CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC1, Ia, HM1.24,HLA-DR, tenascin, VEGF, PIGF, ED-B fibronectin, an oncogene, an oncogeneproduct, NCA 66a-d, necrosis antigens, Ii, IL-2, T101, TAC, IL-6,TRAIL-R1 (DR4) and TRAIL-R2 (DR5) with one or more immunoconjugates ofanti-CD19, or antibodies to theses recited antigens, conjugated withtherapeutic agents, including drugs, toxins, immunomodulators, hormones,enzymes, oligonucleotides, therapeutic radionuclides, etc., with one ormore therapeutic agents, including drugs, toxins, enzymes,oligonucleotides, immunomodulators, hormones, therapeutic radionuclides,etc., administered concurrently or sequentially or according to aprescribed dosing regimen, with the MAbs. Preferred B-cell antigensinclude those equivalent to human CDI9, CD20, CD21, CD22, CD23, CD46,CD52, CD74, CD80, and CD5 antigens. Preferred T-cell antigens includethose equivalent to human CD4, CD8 and CD25 (the IL-2 receptor)antigens. An equivalent to HLA-DR antigen can be used in treatment ofboth B-cell and T-cell disorders. Particularly preferred B-cell antigensare those equivalent to human CD19, CD20, CD22, CD21, CD23, CD74, CD80,and HLA-DR antigens. Particularly preferred T-cell antigens are thoseequivalent to human CD4, CD8 and CD25 antigens. CD46 is an antigen onthe surface of cancer cells that block complement-dependent lysis (CDC).

Further, the present invention contemplates the administration of animmunoconjugate for diagnostic and therapeutic uses in B cell lymphomasand other disease or disorders. An immunoconjugate, as described herein,is a molecule comprising an antibody component and a therapeutic ordiagnostic agent, including a peptide which may bear the diagnostic ortherapeutic agent. An immunoconjugate retains the immunoreactivity ofthe antibody component, i.e., the antibody moiety has about the same orslightly reduced ability to bind the cognate antigen after conjugationas before conjugation.

A wide variety of diagnostic and therapeutic reagents can beadvantageously conjugated to the antibodies of the invention. Thetherapeutic agents recited here are those agents that also are usefulfor administration separately with the naked antibody as describedabove. Therapeutic agents include, for example, chemotherapeutic drugssuch as vinca alkaloids, anthracyclines, epidophyllotoxins, taxanes,antimetabolites, alkylating agents, antibiotics, COX-2 inhibitors,antimitotics, antiangiogenic and apoptotoic agents, particularlydoxorubicin, methotrexate, taxol, CPT-11, camptothecans, proteosomeinhibitors, and others from these and other classes of anticanceragents, thalidomide and derivates, oligonucleotides, particularlyantisense and RNAi oligonucleotides (e.g., against bcl-2), and the like.Other useful cancer chemotherapeutic drugs for the preparation ofimmunoconjugates and antibody fusion proteins include nitrogen mustards,alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2inhibitors, pyrimidine analogs, purine analogs, platinum coordinationcomplexes, enzymes, hormones, and the like. Suitable chemotherapeuticagents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed.(Mack Publishing Co. 1995), and in GOODMAN AND GILMAN'S THEPHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co.1985), as well as revised editions of these publications. Other suitablechemotherapeutic agents, such as experimental drugs, are known to thoseof skill in the art.

Additionally, a chelator such as DTPA, DOTA, TETA, or NOTA or a suitablepeptide, to which a detectable label, such as a fluorescent molecule, orcytotoxic agent, such as a heavy metal or radionuclide, can beconjugated. For example, a therapeutically useful immunoconjugate can beobtained by conjugating a photoactive agent or dye to an antibodycomposite. Fluorescent compositions, such as fluorochrome, and otherchromogens, or dyes, such as porphyrins sensitive to visible light, havebeen used to detect and to treat lesions by directing the suitable lightto the lesion. In therapy, this has been termed photoradiation,phototherapy, or photodynamic therapy (Jori et al. (eds.), PHOTODYNAMICTHERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van denBergh, Chem. Britain 22:430 (1986)). Moreover, monoclonal antibodieshave been coupled with photoactivated dyes for achieving phototherapy.Mew et al., J. Immunoi. 130:1473 (1983); idem., Cancer Res. 45:4380(1985); Oseroff et al., Proc. Natl. Acad. Sci. USA 83:8744 (1986);idem., Photochem. Photohiol. 46:83 (1987); Hasan et al., Prog. Clin.Biol. Res. 288:471 (1989); Tatsuta et al., Lasers Surg Med. 9:422(1989); Pelegrin et al., Cancer 67:2529 (1991). However, these earlierstudies did not include use of endoscopic therapy applications,especially with the use of antibody fragments or subfragments. Thus, thepresent invention contemplates the therapeutic use of immunoconjugatescomprising photo active agents or dyes.

Also contemplated by the present invention are the use of radioactiveand non-radioactive agents as diagnostic agents. A suitablenon-radioactive diagnostic agent is a contrast agent suitable formagnetic resonance imaging, computed tomography or ultrasound. Magneticimaging agents include, for example, non-radioactive metals, such asmanganese, iron and gadolinium, complexed with metal-chelatecombinations that include 2-benzyl-DTP A and its monomethyl andcyclohexyl analogs, when used along with the antibodies of theinvention. See U.S. Ser. No. 09/921,290 filed on Oct. 10, 2001, which isincorporated in its entirety by reference.

Furthermore, a radiolabeled antibody or immunoconjugate may comprise agamma-emitting radioisotope or a positron-emitter useful for diagnosticimaging. Suitable radioisotopes, particularly in the energy range of 60to 4,000 keV, include ¹³¹I, ¹²³I, ¹²⁴I, ⁸⁶Y, ⁶²Cu, ⁶⁴Cu, ¹¹¹In, ⁶⁷Ga,⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹⁸F, ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br, and the like. Seefor example, U.S. patent application entitled “Labeling Targeting Agentswith Gallium-68”—Inventors G. L. Griffiths and W. J. McBride, (U.S.Provisional Application No. 60/342,104), which discloses positronemitters, such as ¹⁸F, ⁶⁸Ga, ^(94m)Tc. and the like, for imagingpurposes and which is incorporated in its entirety by reference.

A toxin, such as Pseudomonas exotoxin, may also be complexed to or formthe therapeutic agent portion of an antibody fusion protein of ananti-CD19 antibody of the present invention. Other toxins suitablyemployed in the preparation of such conjugates or other fusion proteins,include ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example,Pastan et al., Cell 47:641 (1986), and Goldenberg, C A—A Cancer Journalfor Clinicians 44:43 (1994). Additional toxins suitable for use in thepresent invention are known to those of skill in the art and aredisclosed in U.S. Pat. No. 6,077,499, which is incorporated in itsentirety by reference.

An immunomodulator, such as a cytokine may also be conjugated to, orform the therapeutic agent portion of an antibody fusion protein or beadministered with the humanized anti-CD19 antibodies of the presentinvention. Suitable cytokines for the present invention include, but arenot limited to, interferons and interleukins, as described below.

9. Preparation of Immunoconjugates

Any of the antibodies or antibody fusion proteins of the presentinvention can be conjugated with one or more therapeutic or diagnosticagents. Generally, one therapeutic or diagnostic agent is attached toeach antibody or antibody fragment but more than one therapeutic agentor diagnostic agent can be attached to the same antibody or antibodyfragment. The antibody fusion proteins of the present invention comprisetwo or more antibodies or fragments thereof and each of the antibodiesthat compose this fusion protein can contain a therapeutic agent ordiagnostic agent. Additionally, one or more of the antibodies of theantibody fusion protein can have more than one therapeutic of diagnosticagent attached. Further, the therapeutic agents do not need to be thesame but can be different therapeutic agents. For example, one canattach a drug and a radioisotope to the same fusion protein.Particularly, an IgG can be radiolabeled with ¹³¹I and attached to adrug. The ¹³¹I can be incorporated into the tyrosine of the IgG and thedrug attached to the epsilon amino group of the IgG lysines. Boththerapeutic and diagnostic agents also can be attached to reduced SHgroups and to the carbohydrate side chains.

Bispecific antibodies of the present invention are useful inpretargeting methods and provide a preferred way to deliver twotherapeutic agents or two diagnostic agents to a subject. U.S. Ser. No.09/382,186 discloses a method of pretargeting using a bispecificantibody, in which the bispecific antibody is labeled with ¹²⁵I anddelivered to a subject, followed by a divalent peptide labeled with^(99m)Tc. U.S. Ser. Nos. 09/382,186 and 09/337,756 discloses a method ofpre targeting using a bispecific antibody, in which the bispecificantibody is labeled with ¹²⁵I and delivered to a subject, followed by adivalent peptide labeled with ^(99m)Tc, and are incorporated herein byreference in their entirety. Pretargeting methods are also described inU.S. Ser. No. 09/823,746 (Hansen et al.) and Ser. No. 10/150,654(Goldenberg et al.), and US Provisional Application filed Jan. 31, 2003,entitled “Methods and Compositions for Administration of Therapeutic andDiagnostic Agents, Atty Docket No. 018733/1103 (McBride et al.), whichare all also incorporated herein by reference in their entirety. Thedelivery results in excellent tumor/normal tissue ratios for ¹²⁵I and^(99m)Tc, thus showing the utility of two diagnostic radioisotopes. Anycombination of known therapeutic agents or diagnostic agents can be usedto label the antibodies and antibody fusion proteins. The bindingspecificity of the antibody component of the MAb conjugate, the efficacyof the therapeutic agent or diagnostic agent and the effector activityof the Fc portion of the antibody can be determined by standard testingof the conjugates.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation. As analternative, such peptides can be attached to the antibody componentusing a heterobifunctional crosslinker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the therapeutic ordiagnostic agent can be conjugated via a carbohydrate moiety in the Fcregion of the antibody. The carbohydrate group can be used to increasethe loading of the same peptide that is bound to a thiol group, or thecarbohydrate moiety can be used to bind a different peptide.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, all of which are incorporated in their entirety by reference.The general method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality of peptide.This reaction results in an initial Schiff base (imine) linkage, whichcan be stabilized by reduction to a secondary amine to form the finalconjugate.

The Fc region is absent if the antibody used as the antibody componentof the immunoconjugate is an antibody fragment. However, it is possibleto introduce a carbohydrate moiety into the light chain variable regionof a full length antibody or antibody fragment. See, for example, Leunget al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S. Pat. No.5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868, all of whichare incorporated in their entirety by reference. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

10. Pharmaceutically Acceptable Excipients

The humanized, chimeric and human anti-CD19 MAbs to be delivered to asubject can consist of the MAb alone, immunoconjugate, fusion protein,or can comprise one or more pharmaceutically suitable excipients, one ormore additional ingredients, or some combination of these.

The immunoconjugate or naked antibody of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the immunoconjugate or naked antibody are combinedin a mixture with a pharmaceutically suitable excipient. Sterilephosphate-buffered saline is one example of a pharmaceutically suitableexcipient. Other suitable excipients are well-known to those in the art.See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUGDELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

The immunoconjugate or naked antibody of the present invention can beformulated for intravenous administration via, for example, bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic or diagnostic conjugate or nakedantibody. Control release preparations can be prepared through the useof polymers to complex or adsorb the immunoconjugate or naked antibody.For example, biocompatible polymers include matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et al.,Bio/Technology 10: 1446 (1992). The rate of release of animmunoconjugate or antibody from such a matrix depends upon themolecular weight of the immunoconjugate or antibody, the amount ofimmunoconjugate, antibody within the matrix, and the size of dispersedparticles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al.,supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The immunoconjugate, antibody fusion proteins, or naked antibody mayalso be administered to a mammal subcutaneously or even by otherparenteral routes. Moreover, the administration may be by continuousinfusion or by single or multiple boluses. In general, the dosage of anadministered immunoconjugate, fusion protein or naked antibody forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. Typically, it is desirable to provide the recipient with adosage of immunoconjugate, antibody fusion protein or naked antibodythat is in the range of from about 1 mg/kg to 20 mg/kg as a singleintravenous infusion, although a lower or higher dosage also may beadministered as circumstances dictate. This dosage may be repeated asneeded, for example, once per week for 4-10 weeks, preferably once perweek for 8 weeks, and more preferably, once per week for 4 weeks. It mayalso be given less frequently, such as every other week for severalmonths. The dosage may be given through various parenteral routes, withappropriate adjustment of the dose and schedule.

For purposes of therapy, the immunoconjugate, fusion protein, or nakedantibody is administered to a mammal in a therapeutically effectiveamount. A suitable subject for the present invention are usually ahuman, although a non-human animal subject is also contemplated. Anantibody preparation is said to be administered in a “therapeuticallyeffective amount” if the amount administered is physiologicallysignificant. An agent is physiologically significant if its presenceresults in a detectable change in the physiology of a recipient mammal.In particular, an antibody preparation of the present invention isphysiologically significant if its presence invokes an antitumorresponse or mitigates the signs and symptoms of an autoimmune diseasestate. A physiologically significant effect could also be the evocationof a humoral and/or cellular immune response in the recipient mammal.

11. Methods of Treatment

The present invention contemplates the use of naked anti-CD19 antibodiesof the present invention as the primary composition for treatment of Bcell disorders and other diseases. In particular, the compositionsdescribed herein are particularly useful for treatment of variousautoimmune as well as indolent forms of B-cell lymphomas, aggressiveforms of B-cell lymphomas, chronic lymphatic leukemias, acute lymphaticleukemias, and Waldenstrom's macroglobulinemia. For example, thehumanized anti-CD19 antibody components and immunoconjugates can be usedto treat both indolent and aggressive forms of non-Hodgkin's lymphoma.

As discussed above, the antibodies of the present invention are alsosuitable for diagnosis and treatment of various autoimmune diseases.Such diseases include acute idiopathic thrombocytopenic purpura, chronicidiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetesmellitus, Henoch-Schonlein purpura, post-streptococcalnephritis,erythema nodosurn, Takayasu's arteritis, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitisubiterans, Sjogren'ssyndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, parmphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia,perniciousanemia, rapidly progressive glomerulonephritis, psoriasis, andfibrosing alveolitis. The most common treatments are corticosteroids andcytotoxic drugs, which can be very toxic. These drugs also suppress theentire immune system, can result in serious infection, and have adverseaffects on the bone marrow, liver and kidneys. Other therapeutics thathave been used to treat Class III autoimmune diseases to date have beendirected against T-cells and macrophages. There is a need for moreeffective methods of treating autoimmune diseases, particularly Classill autoimmune diseases.

The compositions for treatment contain at least one humanized, chimericor human monoclonal anti-CD19 antibody alone or in combination withother antibodies, such as other humanized, chimeric, or humanantibodies, therapeutic agents or immunomodulators. In particular,combination therapy with a fully human antibody is also contemplated andis produced by the methods as set forth above.

Naked or conjugated antibodies to the same or different epitope orantigen may be also be combined with one or more of the antibodies ofthe present invention. For example, a humanized, chimeric or human nakedanti-CD19 antibody may be combined with another naked humanized, nakedchimeric or naked human anti-CD19 (or with a naked CD20, CD22 or otherB-cell lineage antibody), a humanized, chimeric or human naked anti-CD19antibody may be combined with an anti-CD19 immunoconjugate, a nakedanti-CD19 antibody may be combined with an anti-CD22 radioconjugate oran anti-CD22 naked antibody may be combined with a humanized, chimericor human anti-CD19 antibody conjugated to an isotope, or one or morechemotherapeutic agents, cytokines, toxins or a combination thereof. Afusion protein of a humanized, chimeric or human CD19 antibody and atoxin or immunomodulator, or a fusion protein of at least two differentB-cell antibodies (e.g., a CD19 and a CD22 MAb or a CD19 and a CD20 Mab)may also be used in this invention. Many different antibodycombinations, targeting at least two different antigens associated withB-cell disorders, as listed already above, may be constructed, either asnaked antibodies or as partly naked and partly conjugated with atherapeutic agent or immunomodulator, or merely in combination withanother therapeutic agents, such as a cytotoxic drug or with radiation.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, such as tumor necrosis factor (TNF), andhematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1),IL-2, IL-3, IL-6, IL-I0, IL-12, IL-21 and IL-18), colony stimulatingfactors (e.g., granulocyte-colony stimulating factor (G-CSF) andgranulocyte macrophage-colony stimulating factor (GM-CSF)), interferons(e.g., interferons-alpha, -beta and -gamma), the stem cell growth factordesignated “S1 factor,” erythropoietin and thrombopoietin. Examples ofsuitable immunomodulator moieties include IL-2, IL-6, IL-10, IL-12,IL-18, IL-21, interferon-, TNF-, and the like. Alternatively, subjectscan receive naked anti-CD19 antibodies and a separately administeredcytokine, which can be administered before, concurrently or afteradministration of the naked anti-CD19 antibodies. As discussed supra,the anti-CD19 antibody may also be conjugated to the immunomodulator.The immunomodulator may also be conjugated to a hybrid antibodyconsisting of one or more antibodies binding to different antigens.

Multimodal therapies of the present invention further includeimmunotherapy with naked anti-CD 19 antibodies supplemented withadministration of antibodies that bind CD4, CD5, CD8, CD14, CD15, CD19,CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52,CD54, CD74, CD80, CD126, CD138, B7, MUC1, Ia, HM1.24, HLA-DR (includingthe invariant chain), tenascin, VEGF, PIGF, ED-B fibronectin, anoncogene, an oncogene product, NCA 66a-d, necrosis antigens, Ii, IL-2,T101, TAC, IL-6, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) antibodies in theform of naked antibodies, fusion proteins, or as immunoconjugates. Theseantibodies include polyclonal, monoclonal, chimeric, human or humanizedantibodies that recognize at least one epitope on these antigenicdeterminants. Anti-CD 19 and anti-CD22 antibodies are known to those ofskill in the art. See, for example, Ghetie et al., Cancer Res. 48:2610(1988); Hekman et al., Cancer Immunol. Immunother. 32: 364 (1991);Longo, Curr. Opin. Oncol. 8:353 (1996) and U.S. Pat. Nos. 5,798,554 and6,187,287, incorporated in their entirety by reference. Immunotherapy ofautoimmune disorders with B-cell antibodies is described in the art.See, for example, WO0074718A1, which is incorporated herein by referencein its entirety.

In another form of multimodal therapy, subjects receive naked anti-CD19antibodies, and/or immunoconjugates, in conjunction with standard cancerchemotherapy. For example, “CVB” (1.5 g/m² cyclophosphamide, 200-400mg/m² etoposide, and 150-200 mg/m² carmustine) is a regimen used totreat non-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51: 18(1993). Other suitable combination chemotherapeutic regimens arewell-known to those of skill in the art. See, for example, Freedman etal., “Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME 2, 3rdEdition, Holland et al. (eds.), pages 202&-2068 (Lea & Febiger 1993). Asan illustration, first generation chemotherapeutic regimens fortreatment of intermediate-grade non-Hodgkin's lymphoma (NHL) includeC-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) andCHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Auseful second generation chemotherapeutic regimen is m-BACOD(methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine,dexamethasone and leucovorin), while a suitable third generation regimenis MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine,prednisone, bleomycin and leucovorin). Additional useful drugs includephenyl butyrate and bryostatin-1. Antisense bc1-2 oligonucleotide isalso in clinical trials as a therapeutic for certain malignancies,including B-cell tumors. In a preferred multimodal therapy, bothchemotherapeutic drugs and cytokines are co-administered with anantibody, immunoconjugate or fusion protein according to the presentinvention. The cytokines, chemotherapeutic drugs and antibody orimmunoconjugate can be administered in any order, or together.

In a preferred embodiment, NHL is treated with 4 weekly infusions of thehumanized anti-CD19 antibody at a dose of 200-400 mg/m² weekly for 4consecutive weeks (iv over 2-8 hours), repeated as needed over nextmonths/yrs. Also preferred, NHL is treated with 4 weekly infusions asabove, but combined with epratuzumab (anti-CD22 humanized antibody) onthe same days, at a dose of 360 mg/m2, given as iv infusion over 1 hour,either before, during or after the anti-CD19 monoclonal antibodyinfusion. Still preferred, NHL is treated with 4 weekly infusions of theanti-CD19 antibody as above, combined with one or more injections ofCD22 MAb radiolabeled with a therapeutic isotope such as yttrium-90 (atdose of Y-90 between 5 and 35 mCi/meter-square as one or more injectionsover a period of weeks or months. CD19 MAb may also be combined, insimilar regimens, with anti-CD20 Mabs, such as the hA20 humanized MAb(U.S. application Ser. No. 10/366,709, filed Feb. 14, 2003), whereby aweekly dose×4 weeks per cycle, with optional repeated cycles, is givenof each antibody at an individual dose of 250 mg/m2 i. v. incombination. Either or both antibodies can also be given by s.c.injection, whereby a similar dose is given every other week,particularly for the therapy of patients with autoimmune disease.

In addition, a therapeutic composition of the present invention cancontain a mixture or hybrid molecules of monoclonal naked anti-CD19antibodies directed to different, non-blocking CD19 epitopes.Accordingly, the present invention contemplates therapeutic compositionscomprising a mixture of monoclonal antiCD19 antibodies that bind atleast two CD19 epitopes. Additionally, the therapeutic compositiondescribed herein may contain a mixture of anti-CD 19 antibodies withvarying CDR sequences.

Although naked anti-CD 19 antibodies are the primary therapeuticcompositions for treatment of B cell lymphoma and autoimmune diseases,the efficacy of such antibody therapy can be enhanced by supplementingthe naked antibodies, with supplemental agents, such asimmunomodulators, like interferons, including IFN-alpha, IFN-beta andIFN-gamma, interleukins including IL-1, IL-2, IL-6, IL-12, IL-15, IL-18,IL-21, and cytokines including G-CSF and GM-CSF. Accordingly, the CD19antibodies can be combined not only with antibodies and cytokines,either as mixtures (given separately or in some predetermined dosingregiment) or as conjugates or fusion proteins to the anti-CD19 antibody,but also can be given as a combination with drugs. For example, theanti-CD19 antibody may be combined with CHOP as a 4-drug chemotherapyregimen. Additionally, a naked anti-CD 19 antibody may be combined witha naked anti-CD22 antibodies and/or naked anti-CD20 antibodies and CHOPor Fludarabine as a drug combination for NHL therapy. The supplementaltherapeutic compositions can be administered before, concurrently orafter administration of the anti-CD19 antibodies.

As discussed supra, the antibodies of the present invention can be usedfor treating B cell lymphoma and leukemia, and other B cell diseases ordisorders. The antibodies may be used for treating any disease orsyndrome which involves unwanted or undesirable B-cell activity orproliferation. For example, anti-CD19 antibodies can be used to treatB-cell related autoimmune diseases, including Class III autoimmunediseases such as immune-mediated thrombocytopenias, such as acuteidiopathic thrombocytopenic purpura and chronic idiopathicthrombocytopenic purpura, dermatomyositis, Sjogren's syndrome, multiplesclerosis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, sarcoidosis,ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritisnodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitisubiterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto'sthyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis and fibrosing alveolitis.The antibodies can also be used to treat B-cell diseases such as graftversus host disease, or for transplant immunosuppressive therapy.

Anti-CD19 antibodies may also induce apoptosis in cells expressing theCD19 antigen. Evidence of this induction is supported in the literature.For example, it was demonstrated that apoptosis could be induced usinglymphoid cells that have Fc-receptors reactive with the IgG1-Fc of CD19MAbs that crosslinked. See Shan et al., Cancer Immunol. Immunother.48(12):673-683 (2000). Further, it was reported that aggregates of achimeric CD19 MAb, i.e., homopolymers, induced apoptosis. See Ghetie etal., Blood 97(5): 1392-1398 (2000) and Ghetie et al., Proc. Natl. Acad.Sci USA 94(14): 7509-7514 (1997). Enhancement of the pro-apoptoticactivity of the antibodies may be achieved by simultaneous use of apro-apoptotic agent, such as an agent that inhibits the activity of oneor more members of the anti-apoptosis gene family bcl-2. Antisense andRNAi agents are particularly useful in this regard and can be directedto B cells by conjugation with CD 19 antibodies as described herein.

Antibodies specific to the CD19 surface antigen of B cells can beinjected into a mammalian subject, which then bind to the CD19 cellsurface antigen of both normal and malignant B cells. A mammaliansubject includes humans and domestic animals, including pets, such asdogs and cats. The anti-CD19 MAbs of the present invention, i.e.,humanized, chimeric, human, caninized and felinized, and even murineanti-CD19 MAbs, can be used to treat the non-human maminalian subjectswhen there is a species crossreactivity for the CD 19 antigen. SeeExamples 10 and 11, below. The murine MAbs, which are immunogenic inhumans, are usually less immunogenic in non-human mammalian subjects.The anti-CD19 antibody bound to the CD19 surface antigen leads to thedestruction and depletion of neoplastic B cells. Because both normal andmalignant B cells express the CD19 antigen, the anti-CD19 antibody willresult in B cell death. However, only normal B cells will repopulate andthe malignant B cells will be eradicated or significantly reduced.Additionally, chemical agents or radioactive labels having the potentialto destroy the tumor can be conjugated to the anti-CD 19 antibody suchthat the agent is specifically targeted to the neoplastic B cells.

12. Expression Vectors

The DNA sequence encoding a humanized, chimeric or human anti-CD19 MAbcan be recombinantly engineered into a variety of known host vectorsthat provide for replication of the nucleic acid. These vectors can bedesigned, using known methods, to contain the elements necessary fordirecting transcription, translation, or both, of the nucleic acid in acell to which it is delivered. Known methodology can be used to generateexpression constructs the have a protein-coding sequence operably linkedwith appropriate transcriptional/translational control signals. Thesemethods include in vitro recombinant DNA techniques and synthetictechniques. For example, see Sambrook et al., 1989, MOLECULAR CLONING: ALABORATORY MANUAL, Cold Spring Harbor Laboratory (New York); Ausubel etaI., 1997, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons(New York). Also provided for in this invention is the delivery of apolynucleotide not associated with a vector.

Vectors suitable for use in the instant invention can be viral ornon-viral. Particular examples of viral vectors include adenovirus, AAV,herpes simplex virus, lentivirus, and retrovirus vectors. An example ofa non-viral vector is a plasmid. In a preferred embodiment, the vectoris a plasmid.

An expression vector, as described herein, is a polynucleotidecomprising a gene that is expressed in a host cell. Typically, geneexpression is placed under the control of certain regulatory elements,including constitutive or inducible promoters, tissue-specificregulatory elements, and enhancers. Such a gene is said to be “operablylinked to” the regulatory elements.

Preferably, the expression vector of the instant invention comprises theDNA sequence encoding a humanized, chimeric or human anti-CD19 MAb,which includes both the heavy and the light chain variable and constantregions. However, two expression vectors may be used, with onecomprising the heavy chain variable and constant regions and the othercomprising the light chain variable and constant regions. Stillpreferred, the expression vector further comprises a promoter, a DNAsequence encoding a secretion signal peptide, a genomic sequenceencoding a human IgG1 heavy chain constant region, an Ig enhancerelement and at least one DNA sequence encoding a selection marker.

Also contemplated herein is a method for expressing a humanizedanti-CD19 MAb, comprising (i) linearizing at least one expression vectorcomprising a DNA sequence encoding a humanized, chimeric, or humananti-CD19 MAb, (ii) transfecting mammalian cells with at least one ofsaid linearized vector, (iii) selecting transfected cells which expressa marker gene, and (iv) identifying the cells secreting the humanizedanti-CD 19 MAb from the transfected cells.

13. Methods of Making Anti-CD19 Antibodies

In general, the V_(K) (variable light chain) and V_(H) (variable heavychain) sequences for an anti-CD19 MAb can be obtained by a variety ofmolecular cloning procedures, such as RT-PCR, 5′-RACE, and cDNA libraryscreening. Specifically, the V genes of an anti-CD19 MAb can be clonedby PCR amplification from a cell that expresses a murine or chimericanti-CD19 MAb, sequenced. To confirm their authenticity, the clonedV_(L) and V_(H) genes can be expressed in cell culture as a chimeric Abas described by Orlandi et al., (Proc. Natl. Acad. Sci., USA, 86: 3833(1989)) which is incorporated by reference. Based on the V genesequences, a humanized anti-CD19 MAb can then be designed andconstructed as described by Leung et al. (Mol. Immunol., 32: 1413(1995)), which is incorporated by reference. cDNA can be prepared fromany known hybridoma line or transfected cell line producing a murine orchimeric anti-CD 19 MAb by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2nd Ed(1989)). The V_(K) sequence for the MAb may be amplified using theprimers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primerset described by Leung et al. (BioTechniques, 15: 286 (1993)), which isincorporated by reference, while V_(H) sequences can be amplified usingthe primer pair VH1BACK/VH1FOR (Orlandi et al., 1989 above), or theprimers annealing to the constant region of murine IgG described byLeung et al. (Hybridoma, 13:469 (1994), which is incorporated byreference. The PCR reaction mixtures containing 10

1 of the first strand cDNA product, 10 μl of 1O×PCR buffer [500 mM KCl,100 mM Tris-HCl (pH 8.3), 15 mM MgCl₂, and 0.01% (w/v) gelatin] (PerkinElmer Cetus, Norwalk, Conn.), 250 μM of each dNTP, 200 nM of theprimers, and 5 units of Taq DNA polymerase (Perkin Elmer Cetus) can besubjected to 30 cycles of PCR. Each PCR cycle preferably consists ofdenaturation at 94 C for 1 min, annealing at 50 C for 1.5 min, andpolymerization at 72 C for 1.5 min. Amplified V_(K) and VH fragments canbe purified on 2% agarose (BioRad, Richmond, Calif.). Similarly, thehumanized V genes can be constructed by a combination of longoligonucleotide template syntheses and PCR amplification as described byLeung et al. (Mol. Immunol., 32: 1413 (1995)). See Example 3 for amethod for the synthesis of an oligo A and an oligo B on an automatedRNA/DNA synthesizer (Applied Biosystems, Foster City, Calif.) for use inconstructing humanized V genes.

PCR products for V_(K) can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites to facilitatein-frame ligation of the V_(KK) PCR products. PCR products for V_(H) canbe subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Individual clones containing the respective PCRproducts may be sequenced by, for example, the method of Sanger et al.(Proc. Natl. Acad. Sci., USA, 74: 5463 (1977)), which is incorporated byreference.

The DNA sequences described herein are to be taken as including allalleles, mutants and variants thereof, whether occurring naturally orinduced.

The expression cassettes containing the V_(K) and VH, together with thepromoter and signal peptide sequences can be excised from YKpBR andVHpBS, respectively, by double restriction digestion as HindIII-BamHIfragments. The V_(K) and VH expression cassettes can then be ligatedinto appropriate expression vectors, such as pKh and pG1g, respectively(Leung et al., Hybridoma, 13:469 (1994)). The expression vectors can beco-transfected into an appropriate cell, e.g., myeloma Sp2/0-Ag14 (ATCC,VA), colonies selected for hygromycin resistance, and supernatant fluidsmonitored for production of a chimeric or humanized anti-CD19 MAb by,for example, an ELISA assay, as described below. Alternately, the V_(K)and VH expression cassettes can be assembled in the modified stagingvectors, YKpBR2 and VHpBS2, excised as XbaI/BamHI and XhoI/BamHIfragments, respectively, and subcloned into a single expression vector,such as pdHL2, as described by Gilles et al. (J. Immunol. Methods125:191 (1989) and also shown in Losman et al., Cancer, 80:2660 (1997))for the expression in Sp2/0-Ag14 cells. Another vector that is useful inthe present invention is the GS vector, as described in Barnes et al.,Cytotechnology 32: 109-123 (2000), which is preferably expressed in theNS0 cell line and CHO cells. Other appropriate mammalian expressionsystems are described in Werner et al., Arzneim.-Forsch./Drug Res.48(II), Nr. 8, 870-880 (1998).

Co-transfection and assay for antibody secreting clones by ELISA, can becarried out as follows. About 10 μg of VKpKh (light chain expressionvector) and 20 μg of VHpG1g (heavy chain expression vector) can be usedfor the transfection of 5×10⁶ SP2/0 myeloma cells by electroporation(BioRad, Richmond, Calif.) according to Co et al., J. Immunol., 148:1149 (1992) which is incorporated by reference. Following transfection,cells may be grown in 96-well microtiter plates in complete HSFM medium(Life Technologies, Inc., Grand Island, N.Y.) at 37ΨC, 5% C02. Theselection process can be initiated after two days by the addition ofhygromycin selection medium (Calbiochem, San Diego, Calif.) at a finalconcentration of 500 units/ml of hygromycin. Colonies typically emerge2-3 weeks post-electroporation. The cultures can then be expanded forfurther analysis.

Transfectoma clones that are positive for the secretion of chimeric orhumanized heavy chain can be identified by ELISA assay. Briefly,supernatant samples (˜100 μl) from transfectoma cultures are added intriplicate to ELISA microtiter plates precoated with goat anti-human(GAH)-IgG, F(ab′)₂ fragment-specific antibody (Jackson ImmunoResearch,West Grove, Pa.). Plates are incubated for 1 h at room temperature.Unbound proteins are removed by washing three times with wash buffer(PBS containing 0.05% polysorbate 20). Horseradish peroxidase (HRP)conjugated GAH-IgG, Fc fragment-specific antibodies (JacksonImmunoResearch) are added to the wells, (100 μl of antibody stockdiluted×10⁴, supplemented with the unconjugated antibody to a finalconcentration of 1.0 μg/ml). Following an incubation of 1 h, the platesare washed, typically three times. A reaction solution, [100 μl,containing 167 μg of orthophenylene-diamine (OPD) (Sigma, St. Louis,Mo.), 0.025% hydrogen peroxide in PBS], is added to the wells. Color isallowed to develop in the dark for 30 minutes. The reaction is stoppedby the addition of 50 μl of 4 N HCl solution into each well beforemeasuring absorbance at 490 nm in an automated ELISA reader (Bio-Tekinstruments, Winooski, Vt.). Bound chimeric antibodies are thandetermined relative to an irrelevant chimeric antibody standard(obtainable from Scotgen, Ltd., Edinburg, Scotland).

Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2 μm membrane. The filtered medium is passedthrough a protein A column (1×3 cm) at a flow rate of 1 ml/min. Theresin is then washed with about 10 column volumes of PBS and proteinA-bound antibody is eluted from the column with 0.1 M glycine buffer (pH3.5) containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbance at 280/260 nm. Peak fractions are pooled,dialyzed against PBS, and the antibody concentrated, for example, withthe Centricon 30 (Amicon, Beverly, Mass.). The antibody concentration isdetermined by ELISA, as before, and its concentration adjusted to about1 mg/ml using PBS. Sodium azide, 0.01% (w/v), is conveniently added tothe sample as preservative.

The following are the nucleotide sequences of the primers used toprepare the anti-CD 19 antibodies:

hA19VKA (SEQ ID NO:24) 5′-ATCACTTGTA AGGCCAGCCA AAGTGTTGAT TATGATGGTGATAGTTATTT GAACTGGTAC CAGCAGATTC CAGGGAAAGC ACCTAAATTG TTGATCTACGATGCTTCGAA TCTAGTTTCT GGTATC-3′ hA19VKB (SEQ ID NO:25) 5′-TGCTGACAGTGATATGTTGC AATGTCTTCT GGTTGAAGAG AGCTGATGGT GAAAGTGTAA TCTGTCCCAGATCCGCTGCC AGAGAATCGA GGAGGGATAC CAGAAACTAG ATTCGAAGCA TCGTA-3′hA19VKBack (SEQ ID NO:26) 5′-TCCGACATCC AGCTGACCCA GTCTCCATCA TCTCTGAGCGCATCTGTTGG AGATAGGGTC ACTATCACTT GTAAGGCCAG CCAAAG-3′ hA19VKFor (SEQ IDNO:27) 5′-GCTCCTTGAG ATCTGTAGCT TGGTCCCTCC ACCGAACGTC CACGGATCTTCAGTACTTTG CTGACAGTGA TATGTTGCAA-3′ hA19VHA (SEQ ID NO:28) 5′-CTGGCTACGCTTTCAGTAGC TACTGGATGA ACTGGGTGAG GCAGAGGCCT GGACAGGGTC TTGAGTGGATTGGACAGATT TGGCCTGGAG ATGGTGATAC TAACTACAAT GGAAAGTTCA AGGGGCGCGCCACTATT-3′ hA19VHB (SEQ ID NO:29) 5′-CGTAGTCTCC CGTCTTGCAC AAGAATAGAACGCTGTGTCC TCAGATCGTA GGCTGCTGAG TTCCATGTAG GCTGTATTAG TGGATTCGTCGGCAGTAATA GTGGCGCGCC CCTTGAACTT TCCATTGTA-3′ hA19VHBack (SEQ ID NO:30)5′-CAGGTCCAAC TGCAGCAATC AGGGGCTGAA GTCAAGAAAC CTGGGTCATCG GTGAAGGTCTCCTGCAAGGCT TCTGGCTACG CTTTCAGTAG C-3′ hA19VHFor (SEQ ID NO:31)5′-TGAGGAGACG GTGACCGTGG TCCCTTGGCC CCAGTAGTCC ATAGCATAGT AATAACGGCCTACCGTCGTA GTCTCCCGTC TTGCACAAG-3′

The invention is further described by reference to the followingexamples, which are provided for illustration only. The invention is notlimited to the examples but rather includes all variations that areevident from the teachings provided herein.

EXAMPLE 1 Construction of a Humanized Anti-CD19 Antibody

A chimeric A19 (cA19) antibody was constructed and expressed in Sp2/0cell. The Vk (SEQ ID NO:6) and VH (SEQ ID NO:9) sequences of cA19 areshown in FIG. 1. The cA19 antibody was shown to bound to CD19⁺ humanlymphoma cell lines, such as Raji, Daudi, and Ramos. The Ag-bindingspecificity of purified cA19 was evaluated by a cell surface competitivebinding assay against other anti-CD19 antibodies, e.g. B4 (Coulter) andBU12 (Chembiochem). Briefly, varying concentrations of cA19 wasincubated with Raji cells in the presence of a constant amount of anI-125 radiolabeled anti-CD19 antibody for 1 h. After washing to removethe unbound antibodies, the cell surface-bound radio labeled antibodywas quatitated by counting the cell pellets in a gamma counter. As shownin FIG. 2, cA19 competed with BU12 (Chembiochem) for cell surfacebinding, indicating these antibodies share similar or overlap epitopesof the CD19 molecule.

The light chain and heavy chain variable region sequences encoding thehumanized anti-hCD19 antibody (hA19) were designed and constructed.Comparison of the variable (V) region framework (FR) sequences of thecA19 (FIGS. 1A; SEQ ID NO:1 and SEQ ID NO:2; and 1B; SEQ ID NO:3 and SEQID NO:4) to registered human antibodies in the Kabat database showedthat the FRs of cA19 V_(K) (SEQ ID NO:6) exhibited the highest degree ofsequence homology to that of the human antibody REI (V_(K); SEQ IDNO:5), while the VH sequence was most closely related with that of EU(VH; SEQ ID NO:5). The VH FR4 (SEQ ID NO:11) sequence of the humanantibody, NEWM, however, was better aligned with that of cA19 and usedto replace the EU FR4 sequence for the humanization of the A19 heavychain (FIG. 3B). Therefore, human REI framework sequences were used forVK (FIG. 3A), and a combination of EU and NEWM framework sequences wereused for VH (FIG. 3B). There are a number of amino acid changes in eachchain outside of the CDR regions when compared to the starting humanantibody frameworks. The heavy chain of hA19, hA19VH (SEQ ID NO:10)contains nine changes from the human EU and NEWM frameworks (FIG. 3B).The light chain of Ha19, hA19VK (SEQ ID NO:13), contains nine amino acidchanges from the REI framework (FIG. 2 A). These residues are 4L, 39I,58I, 60P, 87H, 100G, and 107K of V_(K) and 5Q, 27Y, 28A, 40R, 91S, 94R,107T, and 108T of VH. The DNA and amino acid sequences of hA19 V_(K)(SEQ ID NO:12 and SEQ ID NO:13) and VH (SEQ ID NO:14 and SEQ ID NO:15)are shown in FIGS. 4A and 4B, respectively.

EXAMPLE 2 Method of hA19 Antibody Construction

To engineer the CDR-grafted Ha19VH and V_(K) genes, a modified strategyas described by Leung et al.³ was used to construct the designed V_(K)and VH genes for hA19 using a combination of long oligonucleotidesyntheses and PCR. Briefly, two long synthetic oligonucleotides (ca. 130mer in length) representing the 5′-(sense strand, designated as A) and3′-half (anti-sense strand, designated as B) of a V sequence are used asthe templates in a PCR reaction. The 3′-terminal sequences of the longoligonucleotides A and B are designed to be overlap and complementary toeach other. PCR is initiated by annealing of the 3′-termini of A and Bto form a short double strand DNA flanked by the rest of longoligonucleotides (single strand). Each annealed end serves as a primerfor the replication of the single stranded DNA, resulting in elongationof A and B to form the double-strand DNA. In the presence of two shortoligonucleotide primers, V gene segment is generated by PCRamplification of the double strand DNA.

Heavy Chain

For the construction of hA19 VH domain, the long oligonucleotides,hA19VHA (126-mer) and hA19VHB (128-mer) were synthesized on an automatedDNA synthesizer (Applied Biosystem). hA19VHA represents nt 74 to 126 ofthe hA19 VH domain, and hA19VHB represents the minus strand of thehA19VH domain complementary to nt 178 to 306. The 3′-terminal sequences(33 nt residues) of hA19VHA and VHB are complementary to each other. Aminimal amount ofhA19VHA and VHB (determined empirically) was amplifiedin the presence of 10 μL of 10×PCR Buffer (500 mM KCL, 100 mM Tris. HCLbuffer, pH 8.3, 15 mM MgCl.sub.2), 2 μmol of hA19VH Back (5′-CAGGTCCAACTGCAGCAATC AGGGGCTGAA GTCAAGAAAC CTGGGTCATCG GTGAAGGTCTC CTGCAAGGCTTCTGGCTACG CTTTCAGTAG C-3′: SEQ ID NO:30) and hA19VHF or (5′-TGAGGAGACGGTGACCGTGG TCCCTTGGCC CCAGTAGTCC ATAGCATAGT AATAACGGCC TACCGTCGTAGTCTCCCGTC TTGCACAAG-3′: SEQ ID NO:31), and 2.5 units of Taq DNApolymerase (Perkin Elmer Cetus, Norwalk, Conn.). The underlined portionsare the restriction sites for subcloning as shown in FIG. 4B. Thisreaction mixture was subjected to three cycles of polymerase chainreaction (PCR) consisting of denaturation at 94° C. for 1 minute,annealing at 45° C. for 1 minute, and polymerization at 72° C. for 1.5minutes. This procedure was followed by 27 cycles of PCR reactionconsisting of denaturation at 94 C. for 1 minute, annealing at 55° C.for 1 minute, and polymerization at 72° C. for 1 minute. The resultingDNA fragment showed an expected molecular size of 350 in agarose gelelectrophoresis. The double-stranded PCR-amplified product for hA19VHwas gel-purified, restriction-digested with PstI and BstEII restrictionenzymes and cloned into the complementary PstI/BstEII restriction sitesof the heavy chain staging vector, VHpBS2, in which the VH sequence wasfully assembled with the DNA sequence encoding the translationinitiation codon and a secretion signal peptide in-frame ligated at the5′-end and an intron sequence at the 3′-end. VHpBS2 is a modifiedstaging vector of VHpBS (Leung et al., Hybridoma, 13:469 (1994)), intowhich a XhoI restriction site was introduced at sixteen bases upstreamof the translation initiation codon to facilitate the next subcloningstep. The assembled VH gene was subcloned as a XhoI-BamHI restrictionfragment into the expression vector, pdHL2, which contains theexpression cassettes for both human IgG heavy and light chains under thecontrol of IgH enhancer and MT₁ promoter, as well as a mouse dhfr geneas a marker for selection and amplification (FIG. 4B). Since the heavychain region of pdHL2 lacks a BamHI restriction site, this ligationrequires use of a linker to provide a bridge between the BamHI site ofthe variable chain and the HindIII site present in the pdHL2 vector. Theresulting expression vectors were designated as hA19VHpdHL2.

For constructing the full length DNA of the humanized V_(K) sequence,hA19VKA (126-mer, represents nt 61 to 186 of the hA19 V_(K) domain) andhA19VKB (124-mer, represents the minus strand of the hA19 V_(K) domaincomplementary to nt 157 to 281) were synthesized as described above.hA19VKA and VKB were amplified by two short oligonucleotides hA19VK Back(5′- CAGGTCCAAC TGCAGCAATC AGGGGCTGAA GTCAAGAAAC CTGGGTCATCG GTGAAGGTCTCCTGCAAGGCT TCTGGCTACG CTTTCAGTAG C-3′: SEQ ID NO:30′) and hA19VK For(5′-TGAGGAGACG GTGACCGTGG TCCCTTGGCC CCAGTAGTCC ATAGCATAGT AATAACGGCCTACCGTCGTA GTCTCCCGTC TTGCACAAG-3′: SEQ ID NO:31) as described above.The underlined portions are restriction sites for subcloning asdescribed below. Gel-purified PCR products for hA19 V_(K) wererestriction-digested with PvuII and BglII and cloned into thecomplementary PvuII/BclI sites of the light chain staging vector,VKpBR2. VKpBR2 is a modified staging vector of VKpBR (Leung et at,Hybridoma, 13:469 (1994)), into which a XbaI restriction site wasintroduced at sixteen bases upstream of the translation initiationcodon. The assembled V_(K) genes were subcloned as XbaI-BamHIrestriction fragments into the expression vector containing the VHsequence, hA19VHpdHL2. The resulting expression vectors were designatedas hA19pdHL2.

EXAMPLE 3 Transfection and Expression of hA19 Antibodies

Approximately 30 g of the expression vectors for hA19 was linearized bydigestion with SalI and transfected into Sp2/0-Ag14 cells byelectroporation (450V and 25 J-μF). The transfected cells were platedinto 96-well plates for 2 days and then selected for drug-resistance byadding MTX into the medium at a final concentration of 0.075 μM.MTX-resistant colonies emerged in the wells 2-3 weeks. Supernatants fromcolonies surviving selection were screened for human Ab secretion byELISA assay. Briefly, 100 μl supernatants were added into the wells of amicrotiter plate precoated with GAH-IgG, F(ab′)₂ fragment-specific Aband incubated for 1 h at room temperature. Unbound proteins were removedby washing three times with wash buffer (PBS containing 0.05%polysorbate 20). HRP-conjugated GAH-IgG, Fc fragment-specific Ab wasadded to the wells. Following an incubation of 1 h, the plate waswashed. The bound HRP-conjugated Ab was revealed by reading A490 nmafter the addition of a substrate solution containing 4 mM OPD and 0.04%H₂0₂. Positive cell clones were expanded and hB43 were purified fromcell culture supernatant by affinity chromatography on a Protein Acolumn.

EXAMPLE 4 Determination of the Antigen-Binding Specificity and Affinityof antiCD19 Antibodies

The Ag-binding specificity of cA19 and hA190 purified by affinitychromatography on a Protein A column were evaluated and compared by acell surface competitive binding assay. Briefly, a constant amount(100,000 cpm, ˜10 μCi/μg) of ¹²⁵I-labeled cA19 or hA19 was incubatedwith Raji cells in the presence of varying concentrations (0.2-700 nM)of cA19 or hA19 at 4° C. for 1-2 h. Unbound Abs were removed by washingthe cells in PBS. The radioactivity associated with cells was determinedafter washing. As shown in FIG. 2, the purified hA19 competed with¹²⁵I-labeled cA19 for cell surface binding and vice versa, indicatingthe apparent binding avidities are comparable between these two Abs.

The antigen-binding affinity (avidity} constant of hA19 was determinedby direct cell surface binding assay of the radiolabeled Ab andScatchard plot analysis, in comparison to that of cA19. Briefly, hA19and cA19 were labeled with ¹²⁵I by the chloramines-T method. Varyingamounts of either ¹²⁵I-hA19 or ¹²⁵I-cA19 were incubated with 2×10⁵ Rajicells at 4° C. for 2 h and unbound antibodies were removed by washing.The cell-associated radioactivity was counted and Scatchard plotanalysis was performed to determine the maximum number of hA19 and cA19binding sites per cell and the apparent dissociation constants of theequilibrium binding. As shown in FIG. 3, hA19 showed virtually samebinding affinity as cA19. The apparent dissociation constant values forthese two antibodies were calculated to be 1.1 and 1.2 nM, respectively.

EXAMPLE 5 Therapy of Non-Hodgkin's Lymphoma

A patient with indolent, follicular-cell NHL relapses after chemotherapyincluding dexamethasone, and has disease in the chest (para-aortic lymphnodes), an enlarged and involved spleen, and enlarged cervical lymphnodes. The patient is given a course of 300 mg/m² each of humanized CD19MAb of this invention combined with humanized CD20 MAb (hA20)sequentially on the same day by i.v. infusion, weekly for 4 weeks, eachtime being premedicated with Tylenol® and Benadryl® according tostandard, published doses for suppressing infusion-related reactions.Four weeks later, the patient returns for the first follow-upexamination and the only observation is that some of the palpable lymphnodes feel softer. Upon returning 3 months following the first therapycycle, the patient's chest disease appears to have become reduced by 40%on CT scan, the spleen is about half the pre-therapy size, and thecervical lymph nodes are almost gone. The patient is then given aretreatment cycle, and another three months later appears to have anormal-sized spleen, no cervical lymph nodes palpable or measurable onCT scan, and only a small, 1.5-cm lesion in the chest. The patientcontinues to appear almost free-of disease for another 4 months.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention, which isdefined by the following claims.

All of the publications and patent applications and patents cited inthis specification are herein incorporated in their entirety byreference.

1. A method of treating a B-cell disease in a subject comprisingadministering to a subject with a B-cell disease selected from lymphoma,leukemia or autoimmune disease, wherein the B-cells associated with theB-cell disease express CD19 antigen, a therapeutically effective amountof a humanized anti-CD19 antibody or an antigen-binding fragmentthereof, wherein the antibody or antigen-binding fragment thereofcomprises the amino acid sequence of hA19Vk SEQ ID NO:7 and hA19VH SEQID NO:10.
 2. The method of claim 1, wherein the antibody orantigen-binding fragment thereof induces apoptosis of the B-cells thatexpress CD19 antigen.
 3. The method of claim 1, wherein said B-celldisease is selected from the group consisting of B cell lymphoma,non-Hodgkin's lymphoma, chronic lymphocytic leukemia and acutelymphocytic leukemia.
 4. The method of claim 1, further comprisingadministering to said subject concurrently or sequentially atherapeutically effective amount of at least one humanized,chimeric,human or murine Mab.
 5. The method of claim 1, further comprisingadministering to said subject concurrently or sequentially atherapeutically effective amount of at least one therapeutic agent. 6.The method of claim 5, wherein the therapeutic agent is conjugated tothe anti-CD19 antibody or antigen-binding fragment thereof and isadministered concurrently.
 7. The method of claim 5, wherein saidtherapeutic agent comprises a cytotoxic agent, or a radioactive label.8. The method of claim 1, wherein the anti-CD19 antibody orantigen-binding fragment thereof is administered as part of a bispecificor multispecific antibody or antigen-binding fragment thereof, themultispecific or bispecific antibody or antigen-binding fragment thereofcontaining at least one other antibody or antigen-binding fragmentthereof.
 9. The method of claim 8, further comprising administering tosaid subject concurrently or sequentially a therapeutically effectiveamount of at least one therapeutic agent.
 10. The method of claim 7,wherein said cytotoxic agent is a drug or a toxin.
 11. The method ofclaim 5, wherein said therapeutic agent is selected from the groupconsisting of erythropoietin, thrombopoietin, tumor necrosis factor(TNF), an interleukin (IL), granulocyte-colony stimulating factor(G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF)),interferon-alpha, interferon-beta, interferon-gamma, and stem cellgrowth factor S1 factor.
 12. The method of claim 1, wherein said subjectis a human.
 13. The method of claim 5, wherein the therapeutic agent isa chemotherapeutic agent selected from the group consisting of vincaalkaloids, anthracyclines, epidophyllotoxins, taxanes, antimetabolites,alkylating agents, antibiotics, COX-2 inhibitors, antimitotics,doxorubicin, methotrexate, taxol, CPT-11, camptothecans, proteosomeinhibitors, thalidomide, nitrogen mustards, alkyl sulfonates,nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purineanalogs and platinum coordina
 14. A method of inducing apoptosis incells expressing the CD19 antigen comprising exposing cells expressingthe CD19 antigen to a humanized anti-CD19 antibody or an antigen-bindingfragment thereof, wherein the antibody or antigen-binding fragmentthereof comprises the amino acid sequence of hA19Vk SEQ ID NO:7 andhA19VH SEQ ID NO:10.
 15. A method of inducing apootosis in cellsexpressing the CD19 antigen, wherein the cells expressing the CD19antigen are in a subject, comprising administering to the subject ahumanized anti-CD19 antibody or an antigen-binding fragment thereof,wherein the antibody or antigen-binding fragment thereof comprises thesequence of hA19Vk SEQ ID NO:7 and hA19VH SEQ ID NO:10.
 16. The methodof claim 15, wherein the subject has a B-cell disease selected from thegroup onsisting of a B-cell lymphoma, leukemia and autoimmune diseaseand the B-cells associated with the B-cell disease express CD19 antigen.17. The method of claim 16, wherein the B-cell disease is non-Hodgkin'slymphoma, chronic lyitphocytic leukemia or acute lymphocytic leukemia.18. The method of claim 16, further comprising administering to saidsubject concurrently or sequentially a therapeutically effective amountof at least one humanized, chimeric, human or murine Mab.
 19. The methodof claim 16, further comprising administering to said subjectconcurrently or sequentially a therapeutically effective amount of atleast one therapeutic agent.
 20. The method of claim 19, wherein thetherapeutic agent is conjugated tothe anti-CD19 antibody orantigen-binding fragment thereof and is administered concurrently. 21.The method of claim 20, wherein said therapeutic agent comprises acytotoxic agents or a radioactive label.